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Copyright: © 2014 Lynch et al. This is an open-access article distributed under
the terms of the Creative Commons Attribution-NonCommercial-NoDerivs 3.0
Unported License, which permits unrestricted use for non-commercial and educa-
tion purposes only provided the original author and source are credited. The of-
ficial publication credit source: Amphibian & Reptile Conservation at: amphibian-
reptile-conservation. org
Amphibian & Reptiie Conservation 8(1) [Special Sec]: 1-7.
Rediscovery of Andinophryne o/a//a/ Hoogmoed, 1985 (Anura,
Bufonidae), an enigmatic and endangered Andean toad
^Ryan L. Lynch, ^Sebastian Kohn, ^Fernando Ayala- Varela, ^Paul S. Hamilton, and ^Santiago R. Ron
^The Biodiversity Group, Tucson, Arizona, USA ^Rw Manduriacu Cooperative, Quito, ECUADOR ^Museo de Zoologia, Escuela de Biologia,
Pontificia Universidad Catolica del Ecuador, Quito, ECUADOR
Abstract . — ^We report the rediscovery of Andinophryne olaiiai, an endangered species only known
from a single specimen, collected in 1970. At the type locality, Tandayapa, Pichincha Province,
numerous follow-up surveys after 1970 failed to record the species suggesting that the population
is extinct. The rediscovery of A. oiailai took place in 2012 at Ri'o Manduriacu, Imbabura Province,
Ecuador. Two surveys suggest that a healthy population of A. olaiiai survives at the site, with
observations of froglets, juveniles, and adults across numerous stream systems. However, the
extent of known occupancy of the population is small (<1 km^). Further data are presented to update
knowledge of the distribution, ontogeny, morphology, and conservation status of the species. The
population at Ri'o Manduriacu is surrounded by logging, mining, and hydroelectric developments
that could compromise its future survival. There is an urgent need to establish a monitoring program
and to protect its remaining population and habitat in the region.
Key words. Andinophryne olaiiai, rediscovery, Tandayapa Andean toad, Andinosapo de Olalla, Bufonidae, Endan-
gered species, Ecuador
Citation: Lynch RL, Kohn S, Ayala-Varela F, Hamilton PS, Ron SR. 2014. Rediscovery of Andinophryne olaiiai Hoogmoed, 1985 (Anura, Bufonidae), an
enigmatic and endangered Andean toad. Amphibian & Reptile Conservation 8(1) [Special Section]: 1-7 (e75).
Introduction
The small and understudied toad genus Andinophryne
(Bufonidae) is restricted to the western slopes of the
Andes in Colombia and Ecuador. Three species of An-
dinophryne have been described: Andinophryne atelo-
poides (Lynch and Ruiz-Carranza 1981), Andinophryne
colomai (Hoogmoed 1985), and Andinophryne olaiiai
(Hoogmoed 1985). Until recently, all three species were
only known from five or fewer adult individuals at the
type localities: A. atelopoides (Cauca Department, Co-
lombia, 1980), A. colomai (Carchi Province, Ecuador,
1984), and A. olaiiai (Pichincha Province, Ecuador,
1970).
The paucity of information available on Andinophryne
has led to many questions about the taxonomic and con-
servation status of all three species. Andinophryne at-
elopoides, the only species endemic to Colombia and
only known from two specimens, was originally placed
in the genus Bufo by Lynch and Ruiz-Carranza (1981).
Four years later, following the discovery of two similar
bufonid species (A. colomai and A. olaiiai) in northern
Ecuador, and the reexamination of information presented
on B. atelopoides by Lynch and Ruiz-Carranza (1981),
Hoogmoed (1985) created the genus Andinophryne (Bu-
fonidae), and placed all three species within the new ge-
nus.
Despite numerous attempts by trained scientists and
over 150 search hours, subsequent visits to the type lo-
calities of A. colomai and A. olaiiai in Ecuador have
failed to record either species (Coloma et al. 2004; Ron
and Frenkel 2013). Then, in 2005, Murillo et al. (2005)
reported a 160 km range extension for A. olaiiai in Rio
Nambi, Department of Narino, Colombia. This observa-
tion marked the first record of any Andinophryne species
in more than two decades. However, as part of our recent
work with Andinophryne, a member of our team recently
examined a specimen from Rio Nambi and determined
that it was not Andinophryne olaiiai but a different spe-
cies (Santiago Ron, unpubl. data). This identification has
been confirmed by additional fieldwork and specimens
collected at Rio Nambi by Paul David Gutierrez-Carde-
nas (pers. comm.). Therefore, A. olaiiai is the rarest of all
Andinophryne species, with the only known record being
the original type specimen from Tandayapa, Pichincha
Province, Ecuador in 1970.
Forty-three years after the original description of
Andinophryne olaiiai, we report the rediscovery of a
population of A. olaiiai from Rio Manduriacu (herein
Manduriacu), Imbabura Province, Ecuador. We also pro-
Correspondence. ^ryan@ biodiversitygroup.org (corresponding author); ^sebastiankohn® hotmail.com; ^fpayala2000@ gmail.
com; Hamilton @ biodiversity group, org; ^Santiago, r. ron@ gmail. com
Amphib. Reptile Conserv. | amphibian-reptile-conservation.org (1) February 2014 | Volume 8 | Number 1 | e75
Lynch et al.
-a 1,0 'M.O -?9,0 -7?,0
-B1.0 -BO.a -70.0 -78.0 -77.0
Fig. 1. Known records of Andinophryne olallai in northwest
Ecuador; Tandayapa: Type Locality, Manduriacu: New Local-
ity.
vide the first information on the species’ natural history,
geographic range, ontogeny, and conservation status, and
present the first published color photos of live individu-
als across different age classes.
Materials and Methods
Our surveys took place in the premontane tropical for-
est and cloud forests of Manduriacu in NW Ecuador
(1,100-1,400 m), 40 km N of the type locality of A. olal-
lai and near the south border of the Cotacahi-Cayapas
Ecological Reserve (Eig. 1). Surveys were conducted
on 18 November 2012 (original rediscovery) and 13-15
May 2013 using Visual Encounter Surveys (VES) along
stream transects between 19:00 and 01:00 h.
The objectives of the surveys were: (1) determine the
population status of A. olallai', (2) determine the extent
of its occupancy in Manduriacu; and (3) obtain informa-
tion about the behavior and natural history of the species.
Surveys were carried out along small rocky streams with
overhanging herbaceous vegetation (Eig. 2). A total of
three nights were spent surveying four stream systems
neighboring the site of initial discovery (approximately
100 m between streams; < 1 km^ area total).
Information collected in the field included: air temper-
ature (°C), relative humidity (%), time of encounter (24
hr), perch height (cm), snout- vent length (SVL, mm), sex
(when possible), and age class (froglet, juvenile, adult).
Froglets (i.e., recently metamorphosed individuals) were
defined as individuals with heavily patterned dorsum,
lack of pronounced parotoid glands, and SVL between
10-20 mm. Juveniles were defined as individuals with
faint dorsal patterning, more pronounced parotoid glands,
and SVL between 20-30 mm. Adults were defined as
Amphib. Reptile Conserv. | amphibian-reptile-conservation.org (2)
individuals with no dorsal patterning, very pronounced
parotoid glands, presence of large cream- tan colored tu-
bercles on the flanks, and SVL above 30 mm.
Perch height for each individual was measured using
a marked meter stick and SVL measurements were taken
using dial calipers. Climate information was recorded us-
ing a handheld Kestrel 3500 Weather Meter. Individual
toads were only handled when necessary, and always
with use of latex gloves to prevent transferring pathogens
such as amphibian chytrid fungus (Batrachochytrium
dendrobatidis).
Results and Discussion
During the first survey of Manduriacu on 1 8 November
2012, two adult A. olallai were encountered perched on
leaves overhanging a small running stream. Elevation
of the observation site was 1,253 m, and perch heights
of the individuals were 1.5 m and 2.0 m above ground.
Both individuals appeared to be females, based on size,
with SVL of 57 and 58 mm, however sex could not be
determined with complete certainty in the field because
no secondary sexual characteristics are evident in live
Table 1. Reptiles and amphibians associated wiihAndinophyne
olallai at Manduriacu, Imbabura Province, Ecuador and their
current (August 2013) lUCN and EaunaWebEcuador Red List
status (NE - Not Evaluated, DD - Data Deficient, LC - Least
Concern, NT - Near Threatened, V - Vulnerable, EN - En-
dangered). lUCN Red List available at: http://www.iucnredlist.
org/; EaunaWebEcuador Red List available at: http://zoologia.
puce.edu.ee/vertebrados/anfibios/EspeciesEstadoConserva-
cion.aspx.
Species
Fauna Web
Ecuador Red
List
lUCN Red
List
Caecilia guntheri
DD
DD
Centrolene peristictum
NT
V
Epipedobates darwinwallacei
EN
NE
Espadarana prosoblepon
LC
LC
Hyloscirtus alytolylax
NT
NT
Pristimantis achatinus
LC
LC
Pristimantis calcarulatus
LC
V
Pristimantis labiosus
NT
LC
Pristimantis luteolateralis
NT
NT
Pristimantis muricatus
V
V
Pristimantis scolodiscus
DD
EN
Rulyrana orejuela
DD
DD
A lopoglossus festae
NT
NE
Anolis aequatorialis
NT
NE
Anolis gemmosus
LC
LC
Basiliscus galeritus
NE
NE
Bothriechis schlegelii
NT
NE
Cercosaura vertebralis
DD
NE
Diaphorolepis wagneri
NT
NE
Lepidoblepharis conolepis
EN
NE
February 2014 | Volume 8 | Number 1 | e75
Rediscovery of Andinophryne olallai
animals. This initial observation yielded two signifieant
findings: the first evidence of an A. olallai population in
43 years and the second known locality for the species
extending its known range 40 km N from its type locality.
During the course of the survey in May 2013 a total
of 18 A. olallai were observed across four stream sys-
tems. Average nightly environmental conditions during
the three nights of surveys in May were: air temperature
18.3 °C and relative humidity 92.8%. We recorded the
presence of adults, juveniles, and froglets, indicating on-
going population recruitment (Fig. 3). Eleven of the nine-
teen individuals encountered were adults, and although
their sex could not be determined, eggs were visible in
the abdomen of two gravid females. The sex of one pre-
served adult male (QCAZ-A 55561) was confirmed by
internal gonad examination. The confirmed adult females
had SVL of 57 mm and 60 mm, considerably larger than
the SVL reported by Hoogmoed (1985) for the holotype
($, 39.6 mm). The single confirmed male had a SVL of
36.5 mm. Mean SVL for adults with unknown sex was
47.1 mm (n = 8).
All individuals encountered were perched on branch-
es or leaves overhanging or bordering the streams. Mean
perch height was 1.4 m (n = 18), with adults generally
perching higher than younger individuals. Maximum ob-
served perch height was four meters. Although no official
surveys were conducted during the day, no individuals
were observed along streams during random daytime
walks. Although further behavioral work needs to be con-
ducted, this observation suggests that A. olallai may be
actively foraging during the day in the forests surround-
ing streams. At night, they remain immobile perched on
leaves overhanging the streams. Lack of movement may
protect them from predators.
Ontogeny and Morphology
All information on A. olallai reported by Hoogmoed
(1985) was based on two adult specimens. Our obser-
vations of froglets and juveniles mark the first reported
information on the species’ pre-adult morphology and
ontogeny. Ontogenetic change in color pattern is con-
siderable (Fig. 3), and is one of the few reported cases
of such an extreme change in bufonids in Ecuador (see
Hoffman and Blouin 2000). We observed a total of two
froglets (mean SVL 13.1 mm) and five juveniles (mean
SVL 26.6 mm). Eroglets have a copper, gold, and white
dorsum with a mottling pattern reminiscent of some
species of Atelopus (Eig. 3: A, B). This contrasts with
the patternless brown dorsum of the adults. The venter
of froglets have a series of white undulating lines that
extend the length of the body (Eig. 4). The iris in frog-
lets and juveniles is more vibrantly red than in adults,
which have a yellow copper-colored iris that is darker
medially near the horizontally oval pupil. Froglets also
differ from adults in lacking tubercles and parotoid
Fig. 2. Andinophryne olallai habitat from Rio Manduriacu,
Imbabura Province, Ecuador. All individuals encountered were
found perched on branches or leaves along streams similar to
the stream pictured here.
glands. Juveniles retained some of the mottling pattern
seen in froglets (primarily posteriorly on the hind legs)
and lacked the conspicuous tubercles on the flank (Fig.
3: C, D). However, they begin to show adult traits like
pronounced parotoid glands, tan-brown coloration, and
strongly webbed fingers.
Morphological characteristics of the adults match
those of the holotype of A. olallai (comparisons based
on photographs of the holotype, available at Link/URL:
Amphibiaweb Ecuador, and Hoogmoed 1985). The holo-
type and the observed specimens of the population from
Manduriacu differ from the other species of the genus
in having more developed parotoid glands, larger body
size, strongly webbed fingers, and conspicuous yellow-
ish glands scattered on the flanks and arranged in rows
or in irregular patterns (Fig. 3: E, F) (Hoogmoed 1985).
The dorsal texture varies from smooth to mildly tuber-
culate. One individual had abundant tubercles on the
anterior half of the dorsum and large scattered tubercles
on the posterior half. The description of coloration given
by Hoogmoed (1985) was of an animal in preservative;
however, the color description falls within the variation
observed in life at Manduriacu. The only notable differ-
Amphib. Reptile Conserv. | amphibian-reptile-conservation.org (3)
February 2014 | Volume 8 | Number 1 | e75
Lynch et al.
Fig. 3. Ontogenetic transformation of color and pattern in Andinophryne olallai from Rio Manduriacu, Imbabura Province, Ecuador.
(A) Froglet (11 mm SVL; in situ), (B) Froglet (15.1 mm SVL; in situ), (C) Juvenile (26.3 mm SVL; in situ), (D) Juvenile (28.1 mm
SVL; in situ), (E) Adult (44.6 mm SVL; ex situ), (F) Adult (53.3 mm SVL; in situ). Note the progressive ontogenetic change in dor-
sal patterning from heavily mottled to no pattern; lack of parotoid glands and tubercles along the flank to presence of conspicuous
parotoid glands and tubercles along the flank; a darkening of color from copper, tan, and white to dark brown; and iris color change
from vibrant crimson to copper-orange.
Amphib. Reptile Conserv. | amphibian-reptile-conservation.org (4)
February 2014 | Volume 8 | Number 1 | e75
Rediscovery of Andinophryne olallai
Fig. 4. Ventral pattern of froglets of Andinophryne olallai.
Manduriacu, Imbabura Province, Ecuador.
ence is that dorsal and flank coloration is not uniform in
all individuals; the head and dorsum were darker brown
than the light brown-tan flanks in most live animals ob-
served at Manduriacu.
Sympatric Species
During our herpetofaunal surveys of Manduriacu we re-
corded observations of all amphibian and reptile species
occurring at the site (Table 1). Most of these species are
mid-elevation (1,000-2,500 m) inhabitants of premon-
tane and cloud forests of the eastern Andes. A number of
the species (i.e., Lepidoblepharis conolepis, Pristimantis
scolodiscus) are either nationally or internationally listed
as Endangered, and two of the species are categorized
as Data Deficient or have not yet been assessed (i.e., Di-
aphorolepis wagneri, Epipedobates darwinwallacei) and
very little is known about their biology or conservation
status due to few available records or localities.
Conservation and Threats
Andinophryne olallai is currently classified as Data
Deficient by the lUCN Red List (Coloma et al. 2010).
However, more recent assessments considers A. olallai
as Endangered based on its restricted range, the appar-
ent extirpation of the species from the type locality and
Fig. 5. A recently deforested plot of land that is less than one km from the population of Andinophryne olallai in Manduriacu, Im-
babura Province, Ecuador.
Amphib. Reptile Conserv. | amphibian-reptile-conservation.org (5)
February 2014 | Volume 8 | Number 1 | e75
Lynch et al.
extensive habitat degradation (Coloma et al. 2011-2012;
Ron and Frenkel 2013). The rarity of known distribution
and a very small population size likely warrants an lUCN
Red List status of Endangered.
Although we found evidence of a seemingly healthy
population of A. olallai at Manduriacu, with the presence
of all size classes across numerous stream systems, the
extent of known occupancy remains extremely small (< 1
km^). At present, pristine habitat still exists at Manduria-
cu, however, the surrounding forest is rapidly disappear-
ing due to a variety of anthropogenic factors (i.e., inten-
sive logging, mining, and hydroelectric development).
These activities are expanding quickly and resulting in
extensive habitat fragmentation and loss (Fig. 5). The
apparent extirpation of A. olallai from the type locality,
a site where forest has been lost and fragmented, sug-
gests that the species is sensitive to anthropogenic habi-
tat change. Urgent conservation measures and population
monitoring are needed in order to ensure the survival of
A. olallai in nature. It is our hope that the rediscovery
of A. olallai will result in immediate support for greater
protection of the forests in and around Manduriacu, and
provide assistance in creating biological corridors be-
tween the neighboring reserves of Los Cedros and Co-
tacachi-Cayapas.
Acknowledgments. — We thank Juan and Monica
Kohn for purchasing and protecting the land at Rio
Manduriacu. Programa Socio Bosque provides support
for conservation of the forests of Manduriacu. Pontifi-
cia Universidad Catolica del Ecuador provided logistical
support for our fieldwork. The Biodiversity Group pro-
vided support for RLL research, and Belisario Cepeda
Quilindo gave access to their 2005 publication on A.
olallai. Paul Gutierrez-Cardenas provided access to
specimens and photographs of A. colomai. This work
was conducted under Ministerio del Ambiente permit #
005-12- IC-EAU-DNB/MA.
Literature Cited
Coloma LA, Guayasamin JM, Menendez-Guerrero P
(editors). 2011-2012. Amphibian Red List of Ecua-
dor, AnfibiosWebEcuador. Otonga Foundation, Quito,
Ecuador.
Coloma LA, Ron SR, Cisneros-Heredia DF, Yanez-Mu-
noz MH, Gutierrez-Cardenas PD, Angulo A. 2004.
Andinophryne olallai. In: lUCN 2011. lUCN Red List
of Threatened Species. Version 2011.2. Available:
http://www.iucnredlist.org. [Accessed: 27 December
2013].
Hoffman EA, Blouin MS. 2000. A review of colour and
pattern polymorphisms in Anurans. Biological Jour-
nal of the Linnean Society 70: 633-665.
Hoogmoed MS. 1985. A new genus of toads (Amphibia:
Anura, Bufonidae) from the Pacific slopes of the An-
des in northern Ecuador and southern Colombia, with
the description of two new species. Zoologische Med-
edelingen 59: 251-274.
Link/URL: Amphibiaweb Ecuador. Available: http://zoo-
logia.puce.edu.ec/vertebrados/anfibios [Accessed: 25
August 2013].
Lynch JD, Ruiz-Carranza PM. 1981. A new species of
toad (Anura: Bufonidae) from the Cordillera Occiden-
tal in southern Colombia. Lozania 33: 1-7.
Murillo Pacheco J, Cepeda Quilindo B, Elorez Pai C.
2005. Andinophryne olallai (Tandayapa Andes toad).
Geographic distribution. Herpetological Review 36:
331.
Ron SR, Frenkel C. 2013. Andinophryne olallai. In: Ron
SR, Guayasamin JM, Yanez-Munoz MH, Merino-
Viteri A (editors). AmphibiaWebEcuador. Version
2013.1. Available: http://zoologia.puce.edu.ee/ver-
tebrados/anfibios/FichaEspecie.aspx?ld=l 140 [Ac-
cessed: 26 July 2013].
Received: 09 December 2013
Accepted: 24 January 2014
Published: 03 February 2014
Ryan L. Lynch is the lead biologist and photographer for Ecuadorian programs for The Biodiversity
Group in Quito, Ecuador. He received his M.S. in wildlife ecology and conservation from the University of
Elorida where he used occupancy modeling to determine the status of anurans across the Elorida everglades
landscape. Ryan’s current research interests focus on the ecology, distribution, and conservation of rare,
threatened, and new species of reptiles and amphibians in Ecuador.
Sebastian Kohn is the administrator for the Antisanilla-Sunfohuaico Reserve run by the Jocotoco Eounda-
tion in Ecuador. He received his B.A. in biology and environmental studies at Whitman College in Wash-
ington State, USA. He currently directs the Rio Manduriaco Cooperative in Imbabura, Ecuador, as well as
the llitio Wildlife Rescue Center and Hacienda llitio in Cotopaxi, Ecuador. Sebastian is a founding member
of the Andean Condor Conservation Group of Ecuador (Grupo Nacional de Trabajo del Condor Andino)
and has been working with, and researching, both wild and captive condors for ten years.
Amphib. Reptile Conserv. | amphibian-reptile-conservation.org (6)
February 2014 | Volume 8 | Number 1 | e75
Rediscovery of Andinophryne olallai
Fernando Ay ala- Varela is the director of the herpetology collection at the Pontificia Universidad Catolica
del Ecuador in Quito. He received his diploma at the Pontificia Universidad Catolica del Ecuador, Quito in
2004. He has been interested in herpetology since childhood and has dedicated a lot of time studying the
lizards of Ecuador, specifically the taxonomy and ecology of Anolis species. His current research interests
include reproductive biology and ecology of lizards and snakes in Ecuador.
Paul S. Hamilton is the founder and executive director of The Biodiversity Group in Tucson, Arizona,
USA. He holds a master’s degree in biology from the University of California, Riverside, and a Ph.D. in
biology from Arizona State University, and has conducted field studies in evolutionary, behavioral and
conservation ecology both in the tropics and the desert southwest. In addition to his research interests in
ecology and conservation of overlooked species such as amphibians, reptiles, and invertebrates, he is also
, __ a well published scientific and artistic photographer.
t Santiago R. Ron is the curator of amphibians and professor at the Pontificia Universidad Catolica del
Ecuador in Quito. His research focuses on the evolution and diversity of neotropical amphibians with
emphasis on Ecuador. Areas covered include evolution of animal communication, sexual selection, sys-
tematics and taxonomy. In the area of conservation biology Santiago is interested in the study of amphibian
extinctions in the Andes. Santiago also oversees the ex situ amphibian conservation project Balsa de los
Sapos at the Pontificia Universidad Catolica del Ecuador in Quito.
Amphib. Reptile Conserv. | amphibian-reptile-conservation.org (7)
February 2014 | Volume 8 | Number 1 | e75
Copyright: © 2014 Ayala- Varela et al. This is an open-access article distributed under
the terms of the Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported
License, which permits unrestricted use for non-commercial and education purposes only
provided the original author and source are credited. The official publication credit source:
Amphibian & Reptile Conservation at: amphibian-reptile-conservation.org
Amphibian & Reptiie Conservation
[Special Section] 8(1) : 8-24.
A new Andean anole species of the Dactyloa clade
(Squamata: Iguanidae) from western Ecuador
Ternando P. Ayala- Varela, ^Diana Troya-Rodn'guez, ^Xiomara Talero-Rodn'guez
and ''Omar Torres-Carvajal
^■^■^■^Escuela de Ciencias Biologicas, Pontificia Universidad Catolica del ECUADOR, Avenida 12 de Octubre 1076 y Roca, Apartado 17-01-2184,
Quito, ECUADOR
Abstract . — ^We describe a new species of Anolis from the western slopes of the Andes of Ecuador,
province of Bolivar. It is referred to (1) the aequatoriaiis series based on its moderate size and
narrow toe lamellae, and (2) the eu/aemus sub-group based on having a typical Ano/is digit, in which
the distal lamellae of phalanx III distinctly overlap the proximal subdigital scales of phalanx II. The
new species is most similar morphologically to A. otongae and A. gemmosus, both from similar
elevations on the western Andean slopes of Ecuador, but differs from these species in morphology
and color patterns. We present a phylogeny based on DNA sequence data as additional evidence
supporting delimitation of the new species. The new species and A. gemmosus are sister taxa
within the “western Dactyloa clade.”
Key words. Clade Dactyloa, DNA, lizard, phylogeny, South America, systematics
Citation: Ayala-Varela FP, Troya-Rodriguez D, Talero-Rodriguez X, Torres-Carvajal O. 2014. A new Andean anole species of the Dactyloa clade (Squa-
mata: Iguanidae) from western Ecuador. Amphibian & Reptile Conservation 8(1) [Special Section]: 8-24 (e76).
Introduction
With nearly 490 described species, anole lizards {Anolis)
have proliferated impressively in the Americas (Nich-
olson 2002; Poe 2004), possibly prompted by ecologi-
cal opportunity (Losos 2009). Although the diversity of
these lizards has been extensively studied in the West
Indies (Losos 2009), the same is not true for the main-
land radiation, which is probably greater than previously
thought. For example, all but two — Anolis ruibali Navar-
ro & Garrido 2004 and 4. sierramaestrae Holahova et al.
2012 — of the 31 new species of Anolis described during
the last decade (2003-2013) occur in mainland Central
and South America (Uetz and Hosek 2014). Improving
knowledge concerning the diversity of mainland anoles
is crucial to understanding the nature of this radiation.
Anole lizards represent the most species-rich clade
traditionally recognized as a genus in Ecuador, with 37
species reported to date (Torres-Carvajal et al. 2014).
The diversity of anole lizards in Ecuador is remarkably
greater west of the Andes, with more than twice the num-
ber of species that occur east of the Andes (25 and 12
species, respectively). Of these, five species have been
described during the last six years from both sides of the
Andes as a result of both careful examination of exist-
ing collections and recent collecting in poorly explored
areas. Here we contribute to that growing body of taxo-
nomic knowledge with the description of a new species
of Anolis endemic to the western slopes of the Andes in
Ecuador. We present molecular evidence supporting rec-
ognition of the new species by performing phylogenetic
analyses of mitochondrial DNA sequence data.
Materials and Methods
Morphological data
All known specimens of the new species described in
this paper are included in the type series, and were de-
posited in the Museo de Zoologia, Pontificia Universi-
dad Catolica del Ecuador, Quito (QCAZ). Specimens of
other species of Anolis examined in this study are listed
in Appendix 1. We follow previously proposed terminol-
ogy (Williams et al. 1995) for measurements and squa-
mation. Nine morphological measurements were taken
with digital calipers and recorded to the nearest 0.1 ncnn:
head length, head width, head height, forelimb length,
hindlimb length, snout- vent length, jaw length, axilla-
groin length, and snout length. In addition, tail length
measurements were taken with a ruler and recorded to
the nearest millimeter; regenerated or broken tails were
not measured. Sex was determined by noting the pres-
ence of hemipenes, which were everted in all male speci-
mens during preparation.
Statistical analyses
Given that the new species is very similar in morphol-
ogy to Anolis gemmosus and A. otongae we performed
Correspondence. fpayala2000@yahoo.com (Corresponding author); ^dianatrl 7@gmail.com; ^xiomy.talero@gmail.com;
^omartorcar@gmail. com
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Ayala- Varela et al.
Fig. 1. Head of the holotype (QCAZ 3449) of Anolis poei sp. nov. in dorsal (top), ventral (middle), and lateral (bottom) views
[Scale bar =10 mm]. Photographs by F Ayala-Varela.
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A new species ofAnolis lizard from western Ecuador
a Principal Component Analysis (PCA) to determinate
whether separation in morphological space between
those species was statistically significant. Principal com-
ponents (PCs) were extracted from a covariance matrix
of the raw and rescaled data. The new species is most
similar to A. gemmosus, for which we also used Mests to
evaluate quantitative differences between both species.
One of the assumptions of the t-test for two samples is
that the variances of both samples are equal: therefore,
F-tests also were performed for each character to test for
equality of variances. If the variances were not the same
(i.e., P < 0.05), an unequal variance t-statistic was used.
Statistical analyses were performed in SPSS Statistics 17
(SPSS Inc. 2008).
The distribution map was prepared in ArcMap 9.3
(ESRI, Inc.); WGS84 is the datum for all coordinates
presented below.
DNA sequence data
Total genomic DNA was digested and extracted from liv-
er or muscle tissue using a guanidinium isothiocyanate
extraction protocol. Tissue samples were first mixed with
Proteinase K and a lysis buffer and digested overnight
prior to extraction. DNA samples were quantified using
a Nanodrop® ND-1000 (NanoDrop Technologies, Inc),
re-suspended and diluted to 25 ng/ul in ddH20 prior to
amplification.
Using primers and amplification protocols from the
literature (Folmer et al. 1994; Kumazawa and Nishida
1993; Macey et al. 1997; Schulte and Cartwright 2009)
we obtained 2807 nucleotides (nt) representing the nucle-
ar gene recombination-activating gene 1 (RAGl, 8 lint),
as well as the mitochondrial genes Cytochrome c oxi-
dase I (COl, 655nt) and a continuous fragment includ-
ing the NADH dehydrogenase subunit 2 (ND2, 1038 nt),
tRNATrp, tRNAAla, tRNAAsn, tRNACys (282nt), and
the origin of the light-strand replication (Ol, 29nt). The
new sequence data were obtained for three individuals of
the new species described herein, two of A. gemmosus,
and two of otongae. In addition we used sequence data
generated by Castaneda and de Queiroz (2011) for 20 in-
dividuals of the clade Dactyloa, as well as one sequence
of A. occultus, which was used as the outgroup in the
phylogenetic analysis. Gene regions of taxa included in
phylogenetic analyses along with their GenBank acces-
sion numbers are shown in Table 1.
Phylogenetic analyses
Editing, assembly, and alignment of sequences were
performed with Geneious ProTM 5.3 (Biomatters Ltd.
2010). Genes were combined into a single dataset with
eleven partitions, three per protein coding gene corre-
sponding to each codon position, one with all tRNAs, and
one with the Ol. The best partition strategy along with the
corresponding models of evolution were obtained in Par-
titionFinder 1.1.1 (Lanfear et al. 2012) under the Bayes-
ian information criterion.
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Phylogenetic relationships were assessed under a
Bayesian approach in MrBayes 3.2.0 (Ronquist and
Huelsenbeck 2003). Four independent analyses were
performed to reduce the chance of converging on a lo-
cal optimum. Each analysis consisted of 20 million
generations and four Markov chains with default heat-
ing values. Trees were sampled every 1,000 generations
resulting in 20,000 saved trees per analysis. Stationarity
was confirmed by plotting the -In L per generation in the
program Tracer 1.6 (Rambaut et al. 2013). Additionally,
the standard deviation of the partition frequencies and the
potential scale reduction factor (Gelman and Rubin 1992)
were used as convergence diagnostics for the posterior
probabilities of bipartitions and branch lengths, respec-
tively. Adequacy of mixing was assessed by examining
the acceptance rates for the parameters in MrBayes and
the effective sample sizes (ESS) in Tracer. After analyz-
ing convergence and mixing, 2,000 trees were discarded
as “bum-in” from each mn. We then confirmed that the
four analyses reached stationarity at a similar likelihood
score and that the topologies were similar, and used the
resultant 72,000 trees to calculate posterior probabilities
(PP) for each bipartition on a 50% majority mle consen-
sus tree.
Systematics
The taxonomic conclusions of this study are based on the
observation of morphological features and color patterns,
as well as inferred phylogenetic relationships. We con-
sider this information as species delimitation criteria fol-
lowing the general species concept of de Queiroz (1998,
2007).
Anolis poei sp. nov.
urn:lsid:zoobank.org:act:712687F6-CF33-4969-815D-E4600D01FB4C
Proposed standard English name: Telimbela anoles
Proposed standard Spanish name: Anolis de Telimbela
Holotype
QCAZ 3449 (Figs. 1, 2), adult male, Ecuador, Provincia
Bolivar, Telimbela, 01.65789°S, 79.15334°W, WGS84
1,354 m, 10 June 2011, collected by Fernando Ayala- Va-
rela, Jorge H. Valencia, Diana Troya-Rodriguez, Francy
Mora, and Estefama Boada.
Paratypes (1 5)
ECUADOR: Provincia Bolivar: QCAZ 3444-3448,
3451-3455, 4359, same data as holotype, ex-
cept 0.1658440°S, 79.157150°W, 1,310 m; QCAZ
6781-6783 Telimbela, Escuela Elisa Marino de Carva-
jal, 0.1665857°S, 79.172096°W, 27 July 2004, collected
by Edwin Carrillo-Ponce and Morley Read; QCAZ 9219
Guaranda, Salinas, Recinto Tres Cmces, 01.431380°S,
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Ayala- Varela et al.
Fig. 2. Anolis poei sp. nov. Holotype, adult male (SVL = 59.67 mm, QCAZ 3449, A), eye close-up (SVL = 60.31 mm, QCAZ 3448,
B), subadult male (SVL =52.12 mm, QCAZ 3455, C, D), adult male (SVL = 59.02 mm, QCAZ 3451, E, F), adult male (SVL =
60.31 mm, QCAZ 3448, G, H). Photographs by L. Bustamante (A), and O. Torres-Carvajal (B, C, D, E, F, G, H).
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A new species ofAnolis lizard from western Ecuador
Fig. 3. Male dewlap of Anolis poei sp. nov. (holotype, QCAZ 3449, A; paratype, QCAZ 3455, B); A. otongae (QCAZ 4661, C;
QCAZ 11791, D); and^. gemmosus (QCAZ 4385, E; QCAZ 4352, F; QCAZ 9452, G; QCAZ 11850, H). Photographs byL. Busta-
mante (A), O. Torres-Carvajal (B, C, D, E, F, H), and S. R. Ron (G).
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Ayala- Varela et al.
Table 1. Species of Anolis sequenced in this study, voucher
specimen numbers, collecting localities, and GenBank acces-
sion numbers.
Species
Voucher
Locality
GenBank Number
A. gemmosus
QCAZ 4385
Ecuador, Car-
chi, Rio San
Pablo near
Chical
ND2: KJ854205
COI: KJ854219
RAGE KJ854212
QCAZ 4406
Ecuador, Car-
chi, Maldo-
nado, Teldibi
Ecological
Trail
ND2: KJ854206
COI: KJ854220
RAGE KJ854213
A. otongae
QCAZ 11790
Ecuador,
Pichincha,
Biological Re-
serve Otonga
ND2: KJ854207
RAGE KJ854214
COI: KJ854221
QCAZ 11791
Ecuador,
Pichincha,
Biological Re-
serve Otonga
ND2: KJ854208
COI: KJ854222
RAGE KJ854215
A. poei
QCAZ 3444
Ecuador,
BoKvar,
Telimbela
ND2: KJ854209
COI: KJ854223
RAGE KJ854216
QCAZ 3445
Ecuador,
BoKvar,
Telimbela
ND2: KJ854210
COI: KJ854224
QCAZ 3448
Ecuador,
BoKvar,
Telimbela
ND2: KJ854211
COI: KJ854225
RAGE KJ854217
QCAZ 4359
Ecuador,
BoKvar,
Telimbela
RAGE KJ854218
79.097970°W, 2,628 m, 28 May 2009, collected by Eli-
cio E. Tapia, Silvia Aldas- Alarcon, and Eduardo Toral-
Contreras.
Diagnosis
We assign Anolis poei both to the aequatorialis series,
based on moderate to large body size, narrow toe lamel-
lae, small head scales, smooth ventral scales, and uni-
form dorsal scalation; and to the eulaemus-subgroup,
based on a typical Anolis digit, in which the distal la-
mellae of phalanx III distinctly overlap the first proximal
subdigital scale of phalanx II (Williams 1976; Williams
and Duellman 1984; Castaneda and de Queiroz 2013).
At present ten species are recognized within the eulae-
mus-subgroup: Anolis anoriensis Velasco et al. 2010,
A. antioquiae Williams 1985, A. eulaemus Boulenger
1908, A. fitchi Williams & Duellman 1984, A. gemmo-
sus O’Shaughnessy 1875, yf. maculigula Williams, 1984,
A. megalopithecus Rueda-Almonacid 1989, A. otongae
Ayala- Varela & Velasco 2010, A. podocarpus Ayala-
Varela & Torres-Carvajal 2010, and A. ventrimaculatus
Boulenger 1911. Anolis poei differs from them mostly
in dewlap features. The dewlap in males of A. poei has a
yellowish-green (or both yellow and green) gorgetal re-
gion, light blue border, and white sternal and marginal
regions (Eig. 3). It has a blackish gorgetal region, and
creamy white sternal region with light brown scales in A.
anoriensis; brown gorgetal region, and pale brown mar-
ginal region in A. eulaemus; bluish-gray gorgetal region,
orange stripes, pale bluish-rose anterior third, and white
sternal region becoming pale blue toward the belly in A.
maculigula; sepia background, with red narrow and ir-
regular stripes on each side of rows in A. megalophitecus;
white, pale yellow, or greenish-yellow gorgetal region,
with white or pale-yellow marginal and sternal regions
in A. otongae (Eig. 3); dull yellowish-green or light blue
gorgetal region, shading to dull cream, greenish yellow
or orange on the marginal region, with white or bluish
green gorgetal rows with or without brown spots and
with yellowish white, yellow or orange sternal region in
A. gemmosus (Eig. 3). The dewlap in males of A. poei
has wide rows of 3-7 scales separated by naked skin; the
width of these rows is one scale in A. fitchi, 2-5 granular,
minute scales in A. podocarpus, 1-2 scales in A. ventri-
maculatus, 3-6 scales in A. otongae, and 2-3 scales in A.
gemmosus. In addition, females of the new species lack
a dewlap, which is present in females of A. anoriensis,
A. antioquiae, A. eulaemus, A. fitchi, and A. podocarpus.
Anolis poei is most similar morphologically to A.
otongae and A. gemmosus (Eig. 4). Erom the former
species (character states in parenthesis) A. poei differs
in having small dorsal chevrons in females (large dorsal
chevrons extending onto flanks), pale yellowish-brown
iris (iris dark blue), interparietal scale (if present) sur-
rounded by small swollen scales (interparietal scale
surrounded by relatively enlarged flat scales), enlarged
postanal scales separated by 3-5 scales (postanal scales
separated by 1-2 scales), and in lacking a dark stripe on
side of head (dark coppery -brown stripe present). Ad-
ditionally, PCA analyses suggested that specimens of A.
poei have shorter jaws, as well as lower and narrower
heads than A. otongae (Table 2, Fig. 5), with PCI (39%
of total variation) represented mainly by head height,
head width, and jaw length.
The new species can be distinguished from A. gem-
mosus (Table 3) in having fewer scales between sec-
ond canthals (11-14, mean = 12.08 and 12-21, mean
= 15.25, respectively; t = 5.31, P<0.005); fewer scales
between supraorbital semicircles (1-3, mean =1.62 and
1-5, mean = 3.13, respectively; t = 4.46, P<0.005); more
lamellae under phalanges III-IV of fourth toe (18-19,
mean = 18.92 and 14-18, mean = 17.33, respectively;
t = -7.86, P<0.005); a narrower head (head width =
7.84-8.84, mean = 8.29 and 6.97-17.41, mean = 10.82,
respectively; t = -7.03, P<0.005); lower head (head
height = 6.54-7.48, mean = 6.92 and 5.42-15.96, mean =
9.51, respectively; t = -6.96, P<0.005); and shorter snout
(snout length = 6.75-7.30, mean = 6.92 and 5.79-14.95,
mean = 10.58, respectively; t = -11.74, P<0.005).
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A new species ofAnolis lizard from western Ecuador
Fig. 4. Part 1. Five species of Anolis from western Ecuador. A. aequatorialis: male (QCAZ 11861, A) and female (QCAZ 3443,
B); A. binotatus: male (QCAZ 3434, C, D); A.fasciatus: male (QCAZ 3450, E, F); A. otongae: male (QCAZ 11790, G) and female
(QCAZ 11791, H).
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Ayala- Varela et al.
Fig. 4. Part 2. A. gemmosus: male (QCAZ 4352, 1, J), male (QCAZ 4385, K, L), male (QCAZ 11849, M, N), and female (QCAZ
4393, O, P). All photographs by O. Torres-Carvajal, except A, M, N (S. R. Ron).
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A new species ofAnolis lizard from western Ecuador
Description of hoiotype (scores for para-
types in parentheses)
Male (Figs. 1, 2); SVL 59.7 mm (46.5-60.3 mm); tail
length 150.2 mm (146.2-163.4 mm); head length 15.9
mm (14.8-16.5 mm); head width 8.4 mm (7. 8-8. 8 mm);
head height 7.2 mm (6. 5-7. 5 mm); internasal distance
2.0 mm (1. 2-2.1 mm); interorbital distance 2.4 mm
(2.2-2. 5 mm); interparietal absent (present, interparietal
length 0.8-0. 9 mm; second largest scale length near in-
terparietal 0. 3-0.4 mm); ear opening maximum length
1.6 mm (1. 6-2.1 mm); snout length 6.8 mm (6. 8-7.3
mm); jaw length 11.7 mm (11.7-14.4 mm); axila-groin
distance 27.7 mm (27.4-30.6 mm); femur length 14.8
mm (14.4-15.6 mm); 4th toe length 12.5 mm (10.6-12.8
mm); 4th toepad width 1.2 mm (1.0- 1.3 mm); forelimb
length 36.2 mm (21.8-36.2 mm); hindlimb length 42.6
mm (42.6-52.7 mm).
Head scales multicarinate (same, unicarinate, or ru-
gose) on frontal region and unicarinate (same, multicari-
nate or rugose) on supraocular disc; 11 (10-14) scales
between second canthals; 13 (11-15) scales between first
canthals; 6 (5-7) scales bordering the rostral posteriorly;
anterior nasal in contact with rostral (same or inferior
nasal in contact with rostral); supraorbital semicircles
separated by two (0-3) scales; supraocular disk with
scales heterogeneous in size; one elongate superciliary
followed by a series of granules (same or one small scale
instead of granules); 6 (5-8) loreal rows on left side; 49
(25-53) loreal scales; interparietal absent (same or, when
present, the interparietal smaller than ear opening, with
4-7 scales between interparietal and semicircles on each
side, and 8-15 scales between interparietal and nape
scales); suboculars in contact with supralabials; 6 (5-7)
supralabials counted up to a point below center of eye;
6 (5-7) infralabials counted up to a point below center
of eye; 7 (4-7) postmentals; one enlarged sublabial on
each side.
Table 2. PCA loadings conducted on nine morphological vari-
ables of Anolis gemmosus, A. otongae and A. poei.
Raw
Rotated
1
2
3
1 2 3
Head height
-0.96
0.21
-0.02
-0.97
0.16
-0.07
Head length
0.24
0.34
0.05
0.22
0.35
0.05
Head width
-0.96
0.20
-0.03
-0.96
0.15
-0.07
Jaw length
0.98
0.06
-0.06
0.98
0.11
-0.03
Snout length
0.82
0.33
-0.07
0.81
0.37
-0.06
Forelimh length
-0.01
0.80
0.04
-0.05
0.80
0.00
Hindlimh length
-0.01
0.85
0.02
-0.05
0.85
-0.03
Axilla-groin length
-0.01
-0.50
-0.02
0.01
-0.50
0.01
Snout- vent length
0.06
-0.04
0.99
0.02
0.01
1.00
Eigenvalue
3.54
1.93
1.00
3.53
1.93
1.01
% van explained
39.31
21.42
11.16
39.23
21.45
11.21
Species
O A. gemmosus
# A (Aongao
G poei
Fig. 5. Distribution of Anolis gemmosus, A. otongae and A. poei
sp. nov. along the first and second principal components axes.
Dorsal crest or enlarged middorsal row absent; dorsal
scales keeled, 11 (9-11) dorsal scales in 5% the length
of SVL contained in the dorsal midline at the level of
the forelimbs; flank scales more or less separated by
skin; ventrals smaller than dorsals, 13 (8-13) longitudi-
nal rows in 5% the length of SVL; ventrals smooth and
granular, arranged in diagonal rows.
Toepads overlap the first phalanx in all toes; 19
(18-19) lamellae under phalanges III and IV of fourth
toe (character 27 in Williams et al. 1995 and character
9 in Poe 2004); supradigitals multicarinate; tail with a
double row of middorsal scales; postanals present (same
or absent), with a slightly enlarged scale laterally on each
side.
Nuchal fold present (absent in females and juveniles);
dorsal folds absent; dewlap extending posteriorly to a
point halfway between fore and hindlimbs (absent in fe-
males); dewlap with five longitudinal rows of 3-7 swol-
len scales, similar size to ventrals, separated by naked
skin.
Sexual variation of meristic and morphometric char-
acters in A. poei is presented in Table 4.
Color in life
Hoiotype (QCAZ 3449; Figs. 2, 3): background of head,
body, limbs and tail green; head with light bluish green,
dark green, and light grey irregular spots dorsally; dor-
sal surface of body with six light grey, small irregular
blotches; dorsal surface of neck with two light grey,
small irregular blotches; limbs with dark green and yel-
lowish-cream spots; lateral surface of head with a white
stripe extending posteriorly from loreal region, through
subocular region, to a point anterior to the tympanum;
white blotch with yellow center above tympanum; eye-
lids yellowish green with first row of upper and lower
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palpebrals black, second and third rows both yellow and
green; lateral surface of neck with dark green dots; body
flanks green, with rows of yellow-centered white spots
oriented ventro-posteriorly; ventral surface of head yel-
lowish green with light yellow blotches; ventral surface
of body white with bluish-green reticulations; ventral
surface of limbs white with several transparent scales
and dark brown reticulations; ventral surface of tail white
with dark green spots anteriorly, and yellowish-green
transverse bands posteriorly; dewlap skin light blue, dark
yellowish green on gorgetal region, light blue on ster-
nal region; gorgetal scales light yellowish green; mar-
ginals and stemals white; iris dark brown with a white
inner ring. When stressed, the dorsal background color
switched from green to yellowish brown.
Subadult male (QCAZ 3455, Figs. 2,3, differences
from holotype): head with dark green and white irregu-
lar small spots dorsally; dorsal surface of body and neck
with white and dark green small spots, and larger pale
yellow spots; lateral surface of head yellowish green with
a white stripe extending posteriorly from loreal region,
through subocular region, to upper border of tympanum;
lateral surface of body with rows of white and dark green
small spots, and larger pale yellow spots; ventral surface
of head with white blotches and light blue spots; ventral
surface of body with dark green reticulations; ventral sur-
face of limbs with brown or green reticulations; ventral
surface of tail with blackish green reticulations anteri-
orly; dewlap skin white, yellow on gorgetal region, white
on sternal region; throat, edge of mouth, and tongue pink-
ish white (Fig. 6). When stressed, rust-colored blotches
appeared on dorsal surface of head, body, limbs and tail.
Adult female (QCAZ 3454, Fig. 7): dorsal surface
of head, body and tail yellowish green; dorsal surface
of body with six narrow brown chevrons, each one de-
limited posteriorly by a grayish white blotch; limbs yel-
lowish green with dark green spots arranged in bands,
and pale yellowish spots; tail with two brown chevrons
anteriorly; lateral surface of head yellowish green; loreal
region yellow; lateral surface of neck and body yellowish
green with brown dots; ventral surface of head pale yel-
low with yellowish green reticulations, short white lon-
gitudinal stripe on throat; ventral surface of body and tail
white with black reticulations laterally; ventral surface
of limbs white with some transparent scales and brown
reticulations on hindlimbs; ventral surface of tail with
brownish green reticulations anteriorly; iris brown with
a pale white ring.
Subadult female (QCAZ 3446, Fig. 7, differences with
QCAZ 3454): occipital and temporal regions with brown
and white small blotches; dorsal surface of neck with a
distinct brown chevron delimited posteriorly by a grayish
white blotch; lateral surface of body yellowish green dor-
sally and light blue ventrally, with white or cream spots;
dorsal surface of tail with two brown chevrons, each one
delimited posteriorly by a grayish white blotch.
Fig. 6. Tongue of Anolis poei sp. nov., subadult male (QCAZ
3455, top); A. gemmosus, adult male (QCAZ 4347, middle); A.
otongae, adult male (QCAZ 4661, bottom). Photographs by S.
R. Ron (top), O. Torres-Carvajal (middle, bottom).
Color in preservative
Holotype (QCAZ 3449): dorsal background of head,
body, limbs and tail grayish brown; dorsal surface of
head with metallic green, dark green, blue, gray and
white cream irregular spots; dorsal surface of body with
six black small chevrons, each delimited posteriorly by a
white irregular blotch; limbs with dark brown and white
spots; lateral surface of head with a white stripe extend-
ing posteriorly from loreal region, through subocular re-
gion, to a point anterior to the tympanum; upper border
of tympanum with a white spot; eyelids purple with first
row of upper and lower palpebrals black, second and
third rows white and purple; neck flanks with black dots;
body flanks grayish brown, with dark brown diagonal
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A new species ofAnolis lizard from western Ecuador
Table 3. Summary of morphological characters of Anolis poei sp. nov. and A. gemmosus from Ecuador. For each quantitative character, the F-value,
t-value, and corresponding P-values are given. Range and sample size (in parenthesis) followed by mean + standard deviation are given.
Character
A. gemmosus
A. poei sp. nov.
F-value
P
t-value
P
Scales between second canthals
12-21 (24) 15.25 + 1.98
11-14(13) 12.08 + 1.12
2.59
0.12
5.31
<0.005
Postrostrals
5-7 (24) 5.79 + 0.72
5-7 (13)5.92 +0.64
1.37
0.25
-0.55
0.59
Row of loreals
6-10(24)7.25 + 1.15
5-8(13) 6.31 + 1.18
0.03
0.86
2.36
0.02
Scales between supraorbital semicircles
1-5 (24)3.13 + 1.23
1-3 (13) 1.62 + 0.77
5.27
0.03
4.46
<0.005
Scales between interparietal (if present) and
semicircles
3-8 (24) 5.67 + 1.27
4-7(6)5.83 + 1.17
0.18
0.67
-0.29
0.77
Supralabials
5-7 (24) 6.08 + 0.50
5-7(13)6 + 0.41
1.29
0.27
0.51
0.61
Postmentals
4-8 (24) 6.13 + 1.03
4-7(13)5.77 + 0.93
0.18
0.67
1.03
0.31
Lamellae under phalanges III-IV of fourth toe
14-18 (24) 17.33 + 0.92
18-19(13) 18.92 + 0.28
8.71
0.01
-7.86
<0.005
Head length
13.23-18.12 (94) 15.46+ 1.07
14.79-16.5 (7) 15.67 + 0.51
4.67
0.03
0.93
0.37
Head width
6.97-17.41 (94) 10.82 + 3.24
7.84-8.84 (7) 8.29 + 0.36
32.16
<0.005
-7.03
<0.005
Head height
5.42-15.96 (94) 9.51 +3.32
6.54-7.48 (7) 6.92 + 0.38
31.04
<0.005
-6.96
<0.005
Jaw length
7.31-17.43 (94) 12.32 + 3.02
11.73-14.36 (7) 12.44 + 0.91
19.25
<0.005
0.26
0.80
Snout length
5.79-14.95 (94) 10.58 + 2.93
6.75-7.30 (7) 6.92 + 0.19
41.30
<0.005
-11.74
<0.005
Forelimb length
23.41-34.34 (94) 29.43 + 2.28
21.84-36.18 (7) 28.57 + 4.25
0.02
0.89
-0.12
0.90
Hindlimb length
41.51-63.80 (94) 52.82 + 4.13
42.56-52.68 (7) 49.01 + 3.33
1.00
0.32
-2.38
0.02
Axilla-groin length
20.73-33.51 (94) 26.74 + 2.07
27.35-30.61 (7) 28.54+ 1.30
0.95
0.33
2.26
0.03
Snout- vent length
46.71-66.21 (94) 58.34 + 3.65
46.47-60.31 (7) 56.87 + 4.85
0.35
0.56
-1.00
0.32
Tail length
94.94-191 (94) 154.59 + 18.66
146.21-163.37 (7) 154.74 + 6.32
3.82
0.05
0.02
0.98
Table 4. Sexual variation in lepidosis and measurements (mm) of Anolis poei sp. nov. Range followed by mean + standard devia-
tion are given.
Character
Males
Females
n = 4
n = 3
Scales between second canthals
11-13 11.75 + 0.96
12-13 12.67 + 0.58
Postrostrals
5-6 5.75 + 0.5
6-7 6.33 + 0.58
Row of loreals
6-8 7 + 1.15
5-6 5.33 + 0.58
Scales between supraorbital semicircles
1-2 1.75+0.5
1-2 1.67 + 0.577
Scales between interparietal and semicircles
Interparietal absent
6-7 6.50 + 3.78
Supralabials to below center of eye
6
6
Postmentals
4-7 5.25 + 1.5
6-7 6.33 + 0.58
Lamellae under phalanges II-III of fourth toe
19
19
Head length
15.8-16.5 15.95 + 0.38
14.8-15.62 15.29 + 0.44
Head width
7.84-8.84 8.31 +0.41
8.05-8.66 8.26 + 0.34
Head height
6.67-7.48 7.02 + 0.39
6.54-7.27 6.8 + 0.41
Jaw length
11.73-12.65 12.25 + 0.38
11.86-14.36 12.70 + 1.43
Snout length
6.75-7.04 6.87 + 0.12
7.82-7.30 7 + 0.26
Forelimb length
27.94-36.18 30.75 + 3.72
21.84-28.19 25.68 + 3.37
Hindlimb length
42.56-52.68 49.35 + 4.59
47.50-49.56 48.57 + 1.03
Axilla-groin length
27.35-28.17 27.76 + 0.33
27.94-30.61 29.57 + 1.43
Snout- vent length
58.80-60.31 59.45 + 0.68
46.47-58.48 53.43 + 6.22
Tail length
150.20-163.37 157.89 + 5.89
146.21-155.38 150.53 +4.60
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Ayala- Varela et al.
A ^
B
'I7\
f *-
9
c
D
E
F
Fig. 7. Anolis poei sp. nov. Adult female (SVL = 46.47 mm, QCAZ 3454, A, B), subadult female (SVL = 47.99 mm, QCAZ 3446,
C, D), juvenile male (SVL = 26.85 mm, QCAZ 3453, E, F). Photographs by O. Torres-Carvajal
bands oriented ventro-posteriorly and intercalated with Adult male (QCAZ 6783): dorsal surface of head and
white spots; ventral surface of head white with light blue
reticulations; ventral surface of body white with faint
grayish purple reticulations; ventral surface of limbs
grayish cream with dark brown reticulations; ventral sur-
face of tail white anteriorly with a metallic green tint and
grayish purple spots, and gray posteriorly; dewlap skin
with a turquoise gorgetal region and white sternal region;
gorgetal scales light brown with a gold tint internally, and
dark brown externally; dewlap marginals and sternals
white; throat, edge of mouth and tongue white.
body dark brown with gray dots; dorsal surface of limbs
dark brown, with gray dots on forelimbs; lateral surface
of head dark brown with white cream dots dorsal and an-
terior to tympanum; body flanks dark brown with faint
white dots arranged on diagonal lines that reach venter;
ventral surface of head with bluish-purple infralabial
and sublabial regions, and light purple gular region with
white irregular spots; ventral surface of body white with
faint purple reticulations; limbs creamish gray with dark
brown reticulations; ventral surface of tail white with
Amphib. Reptile Conserv. | amphibian-reptile-conservation.org (19) May 2014 | Volume 8 | Number 1 | e76
A new species ofAnolis lizard from western Ecuador
purple mottling anteriorly, and gray posteriorly; dewlap
skin with a light blue gorgetal region and white ster-
nal region; gorgetal scales purple; sternal and marginal
scales white.
Adult female (QCAZ 3454): dorsal surface of head
brown with metallic blue and green frontal and supraocu-
lar regions; dorsal surface of body brown with six nar-
row black chevrons, each one delimited posteriorly by
a white blotch; forelimbs bluish brown with white spots
arranged in stripes; hindlimbs brown with dark brown
bands and dots; tail with two black chevrons anteriorly;
lateral surface of head brown with purple tint; labial re-
gion light purple; lateral aspect of neck and body purple
with black dots; ventral surface of head white with purple
brown stripes; ventral surface of body white with dark
brown dots laterally; ventral surface of limbs grayish
cream with dark brown reticulations on hindlimbs; ven-
tral surface of tail white with dark brown dots.
Subadult female (QCAZ 3446, differences with
QCAZ 3454): occipital and temporal regions with dark
brown, small blotches; dorsal surface of neck with a dis-
tinct dark brown chevron; dorsal surface of body with six
distinct, dark brown chevrons; dorsal surface of tail with
two dark brown chevrons.
Phylogenetic relationships
The data matrix analyzed in this study contained 1,065
unique site patterns. Of the 2,807 nucleotide characters
included in our analysis 1,703 were constant, 224 par-
simony uninformative, and 880 were parsimony infor-
mative. The 50% majority rule consensus tree resulting
from the Bayesian analysis (Fig. 8) is generally congru-
ent with the phytogeny of the clade Dactyloa presented
by Castaneda and de Queiroz (2011). Both the new spe-
cies described here and A. otongae are members of the
aequatorialis series of Castaneda and de Queiroz (2013),
which corresponds roughly to the “western clade” of
Castaneda and de Queiroz (2011). Our phytogeny sup-
ports strongly (PP = 0.99) a sister taxon relationship be-
tween Anolis poei and A. gemmosus, as well as the ex-
clusivity (de Queiroz and Donoghue 1990; de Queiroz
1998) of both species. They form a clade sister (PP =
0.89) to A. otongae. The clade formed by the three spe-
cies is sister (PP = 1) to a clade formed by A. aequatoria-
lis and^. anoriensis.
Distribution and ecology
Anolis poei inhabits low montane evergreen forest (Sierra
1999) on the western slopes of the Andes in central Ecua-
dor, Provincia Bolivar, between 1,310-1,354 m (Fig. 9).
f c u SN M 1 xy 1
A. agasshi KEN2<l04_2
A. iuciae USNM321960
A- transversaUx QCAZ 5 6
^ r A. eiiskiikiTiiiri MBLUZy34
I A. enxkaterriari MBLUZ925
*
Fig. 8. Phytogeny of the “western Dactyloa clade” sensu
Castaneda and de Queiroz (2011), which is part of the ae-
quatorialis series of Castaneda and de Queiroz (2013), and
representatives of the heterodermus series {A. euskalerri-
ari), punctatus series (A. transversalis), roquet series (A. lu-
ciae), latifrons series {A. agassizi), and a. non-Dactyloa Ano-
lis (A. occultus). The tree is a majority rule (50%) consensus
tree of 72,000 trees obtained from a Bayesian analysis of the
mitochondrial genes COl, ND2, and adjacent tRNAs, and
the nuclear gene RAGl. Asterisks correspond to posterior
probability values > 0.99. Voucher information is presented
in Castaneda and de Queiroz (2011) and Table 1.
C
A. p^mcccte QC AZ6&69
A- pcrvccac QC A Z6R79
A fusuiti QCAZ6930
A.L'hhris MRCI26
A. chhris QCAZftR77
A. cftloiis OCA26920
'A. venirifnucitlufns MRC09I
A. venirimciL'nlcitiix MRC1J2
■ A. anqiiatoiiaiis QCAZ(iS55
— A, (tctfuuifn'iijtix QCAZ6SK3
- A. afitiriatixix MiiUAT5l7
r A. cinurKttxfx MHUAl 156S
T- A. anoriensis MHUAT5I6
r A. (itonpae QCAZ] I79<)
1= A. oion^ae QCAZ] i7y]
A. poei QCAZ3445_4359
A. poei QC A Z3444
A. poei QCAZ344S
rl
n.na
4
A - petnnuisiss QCAZ43S5
A. gemmosus QCAZ4406
A. gemmosus QCAZ6X51
A. gemmosus QC A ZtSHB 4
Amphib. Reptile Conserv. | amphibian-reptile-conservation.org (20)
May 2014 | Volume 8 | Number 1 | e76
Ayala- Varela et al.
The new species occurs in sympatry with A. aequa-
torialis, A. binotatus, and A. fasciatus at its type locality
(Fig. 4). Specimens of the new species were collected
along the border of a road, close to rivers, in second-
ary forest, and on shrubs within pastures. All individu-
als were found between 20h00 and 22h00 sleeping with
their heads up, or in a horizontal position on branches or
vines, 0.5^.5 m above ground or streams. The smallest
individual QCAZ 3453 (SVL = 26.9 m; TL = 67.6 mm)
was collected on 11 June 2011.
Etymology
The specific name is a noun in the genitive case and is
a patronym for Steve Poe, who has published important
contributions to the systematics and evolution of Ano-
lis lizards (Poe 2004, 2011). During his collecting trips
to Ecuador in 2009 and 2010, Poe trained several young
herpetologists in field collecting techniques and inspired
them to explore the diversity of anole lizards. This paper
is one of the products resulting from that inspiration.
Acknowledgments. — We thank Jorge H. Valencia,
Francy Mora, and Estefama Boada for assistance in the
field; Santiago Ron and Lucas Bustamante for the pho-
tographs; Paulina Santiana and Andrea Varela for assem-
bling some of the figures; Melissa Rodriguez for helping
with the map. Special thanks to Kevin de Queiroz and
two anonymous reviewers for commenting on previous
versions of this manuscript. OTC received funds from
Secretaria de Educacion Superior, Ciencia, Tecnologia
e Innovacion (SENESCYT). Specimens were collected
under collection permit OOl-IC-FAU/FLO/DRZCHI/MA
and 008-09 IC-EAU-DNB/MA issued by Ministerio de
Ambiente del Ecuador.
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Received: 28 April 2014
Accepted: 20 May 2014
Published: 28 May 2014
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Ayala- Varela et al.
Appendix 1
Additional specimens examined
Anolis gemmosus - Ecuador: Carchr. Chilma Bajo, Finca de Anibal Pozo, 0.86397°N, 78.04723°W, 2,022 m, QCAZ
8681-82; Chilma Bajo, Finca de Anibal Pozo, 0.86495 °N, 78.04979'W, 2,071 m, QCAZ 8683; La Centella, 0.89318°N,
78.13471 'W, 1,800-2,400 m, QCAZ 11784; Maldonado, Sendero Ecologico Teldibi, 0.91301 °N, 78.10782'W, 1,477-
l, 635 m, QCAZ 12272, 12278, QCAZ 12279-80, 4360, 4406, 4408; Rio San Pablo, cerca a Chical, 0.90302°N,
78.1 6284 'W, 1 ,399 m, QCAZ 4377, 4382, 4385-86,4388; Rio San Pablo, cerca a Chical, 0.90327°N, 78.1 6201 °W, 1 ,429
m, QCAZ 4393; Rio Verde and Rio Pablo, Rio Estrellita, Guapil, 1 ,428-1 ,466 m, QCAZ 12289, 12294, 12302; Cotopaxi:
1 1 5 km Qeste de Pilalo, 0.928 °S, 79.057 'W, 1 ,500 m, QCAZ 4072; 1 8.2 km de Quillutuha, via a Pucayacu, 0.67843 °S,
79.01 565 'W, 1 ,420 m, QCAZ 8845-49; Alrededores de San Francisco de Las Pampas, 0.42371 °S, 78.96765 °W, 1 ,800
m, QCAZ 1440-47, 2123; Bosque Integral Qtonga , 0.4194 °S, 79.00345°W, 1,720-2,143 m, QCAZ 2758, 2809-10,
3121, 3126-27, 3131, 3133, 3174, 3180-90, 3863-3866, 3869-71 , 3940, 3974-76, 4028-34, 4224-25, 4657, 4663, 4785,
5060, 5063, 5371, 5477-79, 5482-83, 6770-73, 9888, 10424, 10438-39, 10441-42, 10452, 12057, 12060-65, 12067,
12072-73, 12075, 12077-82, Bosque Integral Qtonga, a lo largo del rio Esmeraldas, 0.46333 °S, 79.05027 °W, QCAZ
7281 -89; Bosque Integral Qtonga, alrededores de la estacion, 0.41 933 °S, 79.00336 'W, 1 ,980 m, QCAZ 1 0697 ; Bosque
Integral Qtonga, arriba de la estacion, 0.41 478 °S, 79.00073 'W, QCAZ 3867-68; Bosque Integral Qtonga, orillas del rio
Esmeraldas, 0.41932°S, 78.99396 'W, 1 ,719 m, QCAZ 10393, 10395, 10399; Bosque Integral Qtonga, sendero a la Es-
tacion, 0.41933 °S, 79.00336 °W, 1 ,646 m, QCAZ 10696; Cerca a Naranjito, 0.41944 °S, 79.00333 'W, QCAZ 7825; San
Francisco de Las Pampas, 0.42371 °S, 78.96765 °W, 1 ,600-1 ,800 m, QCAZ 63, 68-70, 72-79, 3134-53, 3155, 3175; Via
a Qtonga, 0.33183 °S, 78.93791 'W, 1,476-1,700 m, QCAZ 8412; Imbabura: 6 de Julio de Cuellaje, 0.4°N, 78.525 °W,
QCAZ 4346-47; 6 de Julio de Cuellaje, 0.401 07 °N, 78.51 81 'W, 1 ,886 m, QCAZ 4349; 6 de Julio de Cuellaje, 0.401 02°N,
78.51 ZZO'W, 1,897 m, QCAZ 4350; 6 de Julio de Cuellaje, punto 8, 0.4°N, 78.525 'W, QCAZ 4348; 6 de Julio de Cuel-
laje, San Antonio, Cordillera deToisan, 0.45803 °N, 78.54722 'W, QCAZ 9450-53; Carretera nueva via a Cuellaje, Sector
de Santa Clara, Reserva Alto Choco, 0.37603°N, 78.45857°W, 2,062 m, QCAZ 4352-54; La Mina, Junin, 0.2754 °N,
78.6603 °W, 1,715 m, QCAZ 3071; Manduriaco, 0.277 °N, 78.873 °W, 1,330 m, QCAZ 5328; Manduriacu, 7.5 km NE of
Bellavista, 0.31006°N, 78.85757'W, 1,177-1,227 m, QCAZ 11606, 12305-314; 12322, 12324, 12326, 12328, 12331;
Reserva Siempre Verde, NE de Cotacachi, 0.37167°N, 78.421 86 °W, 2,468 m, QCAZ 8837; Reserva Alto Choco, Santa
Rosa, 0.36939 °N, 78.44942'W, 2,109 m, QCAZ 7330-31 ; Pichincha: 1-2 km oeste de Tandayapa, 0.004 °S, 78.663 °W,
2,000 m, QCAZ 2070-71 ; 2.9 km de Tandayapa, 0.00952 °S, 78.65698 'W, 1 ,820 m, QCAZ 406-10; 5 km E Tandayapa,
0.02°S, 78.651 °W, 1,975 m, QCAZ 2066-69; A orillas del Rio Chisinche, en la carretera a Conchacato, 0.448°S,
78.76423 'W, 1,693 m, QCAZ 6884-89; 30 km E de Santo Domingo, hacia la Reserva de Bosque Integral Qtonga,
0.3884 °S, 78.92995 'W, QCAZ 9769-70; 9775; Bosque Protector Mindo - Nambillo, refugio, 0.106°S, 78.687°W, 1,700
m, QCAZ 2910; Cooperative El Porvenir, finca El Cedral, 0.114°N, 78.56993°W, 2,297 m, QCAZ 10501-502; Desviacion
a Mindo, 1-5 km de la interseccion hacia abajo, 0.02853°S, 78.75861 °W, 1,661 m, QCAZ 9724-31; Estacion Cientifica
Rio Guajalito, 0.22676 °S, 78.82171 °W, 1,791-1,814 m, QCAZ 1330, 1333, 1500, 1645, 2682-84, 2786, 2813, 2815-16,
3040-45, 3056-57, 3373, 3385, 4123-25, 4210, 4214, 6413-14, 8859, 8864-65, 9974, 11404, 12088-101; Las Tolas,
0.7281 8 °N, 78.77792 'W, 1,200-1,600 m, QCAZ 11848-49; Manuel Cornejo Astorga (Tandapi), frente a la planta de
agua potable "El Placer" via a Conchacato, 0.42471 °S, 78.78905 'W, 1,500 m, QCAZ 6882; Manuel Cornejo Astorga
(Tandapi), via Atenas a 5 km de la carretera principal, 0.40625 °S, 78.83621 °W, 1 ,671 m, QCAZ 5365-70; Mindo, 1 ,342-
l, 560 m, QCAZ 12350-53, 12356, 12358, 12365, 12370, 12375-76; Mindo Biology Station, 0.07805°S, 78.73194 °W,
QCAZ 7518-20, 7522; Mindo, camino entre Mariposas de Mindo y Mindo Garden, 0.06753°S, 78.7535 'W, 1,361 m,
QCAZ 6851-53, 6858; Mindo Garden, 4 km de Mindo, 0.06901 °S, 78.801 66 °W, QCAZ 2787; Mindo, El Monte, Road
to Mindo Garden, 0.07805 °S, 78.7319°W, QCAZ 7521; Mindo, Sachatamia Lodge, 0.02638 °S, 78.75944 °W, 1,700 m,
QCAZ 11857-59; Nanegalito, Finca El Cedral, 0.1141 °N, 78.57007'W, 2,272 m, QCAZ 9462-63; Pachijal, via Nanegali-
to-Los Bancos, 0.13°S, 78.72644 'W, 1,741 m, QCAZ 5494-500; Palmeras, 0.244 °S, 78.794 °W, 1,800 m, QCAZ 871,
881-83,1351-52, 2244, 3004-06; Recinto Chiriboga, Estacion La Favorite , 0.21307°S, 78.78421 °W, 1,680 m, QCAZ
5383-84; Reserva Ecologica Bosque Nublado "Santa Lucia," 0.11928°N, 78.59647°W, 1,624-1,927 m, QCAZ 10664,
11850-52, 11888-93,11897, 11899; Tandayapa, 0.00591 °N, 78.67455'W, 1,670 m, QCAZ 4086. Locality in error. Pich-
incha, San Antonio de Pichincha, 0.00905 °S, 78.44581 °W, QCAZ 724.
Anolis otongae - Ecuador: Cotopaxi: Alrededores de San Francisco de Las Pampas, 0.42371 °S, 78.96765 'W, 1,800
m, QCAZ 2128; Bosque Integral Qtonga , 0.41944 °S, 79.00333 'W, 1,900-2,300 m, QCAZ 1721 , 2050-52, 3129, 3706,
3796, 3872-73, 4025, 4661, 5481, 6219, 11790-91, 12035, 12056, 12058, 12070-71; Los Libres, QCAZ 2781; Penas
Coloradas, 0.52343°S, 79.05908°W, QCAZ 1696; Pichincha: La Victoria, 0.47747°S, 79.05336'W, 2,104 m, QCAZ
6394-96.
Amphib. Reptile Conserv. | amphibian-reptile-conservation.org (23)
May 2014 | Volume 8 | Number 1 | e76
A new species ofAnolis lizard from western Ecuador
Fernando Ay ala- Varela is the director of the herpetology collection at the Pontificia Universidad Catolica
del Ecuador in Quito. He received his diploma at the Pontificia Universidad Catolica del Ecuador, Quito in
2004. He has been interested in herpetology since childhood and has dedicated a lot of time studying the
lizards of Ecuador, specifically the taxonomy and ecology ofAnolis species. His current research interests
include reproductive biology and ecology of lizards and snakes in Ecuador.
Diana Troya-Rodnguez received a B.Sc. in Biology from Pontificia Universidad Catolica del Ecuador
(PUCE) in 2013. As a student, she joined the Museo de Zoologia QCAZ, Pontificia Universidad Catolica
del Ecuador in Quito, where she developed a great interest in reptiles. She has been studying anole lizards
for the last four years. Eor her undergraduate thesis, Diana worked on the “Comparative phylogeography
of two sympatric species of Anolis (Squamata: Iguanidae) and the impact of global warming on their dis-
tribution.”
Xiomara Talero-Rodnguez is an undergraduate biology student at Pontificia Universidad Catolica del
Ecuador in Quito. She joined Museo de Zoologia QCAZ last year and has been helping with several anole
lizard projects ever since. She is currently interested in studying ecology and behavior of anoles.
Omar Torres-Carvajal graduated in Biological Sciences from Pontificia Universidad Catolica del Ecua-
dor (PUCE) in 1998, and in 2001 received a Master’s degree in Ecology and Evolutionary Biology from
the University of Kansas under the supervision of Dr. Einda Trueb. In 2005 he received a Ph.D. degree
from the same institution with the thesis entitled “Phylogenetic systematics of South American lizards
of the genus Stenocercus (Squamata: Iguania).” Between 2006-2008 he was a postdoctoral fellow at the
Smithsonian Institution, National Museum of Natural History, Washington DC, USA, working under the
supervision of Dr. Kevin de Queiroz. He is currently Curator of Reptiles at the Zoology Museum QCAZ of
PUCE and an Associate Professor at the Department of Biology in the same institution. He has published
more than 30 scientific papers on taxonomy, systematics and biogeography of South American reptiles,
with emphasis on lizards. He is mainly interested in the theory and practice of phylogenetic systematics,
particularly as they relate to the evolutionary biology of lizards.
In accordance with the International Code of Zoological Nomenclature new rules and regulations (ICZN 2012), we have deposited this paper in publicly accessible institutional libraries.
The new species described herein has been registered in ZooBank (Polaszek 2005a, b), the official online registration system for the ICZN. The ZooBank publication LSID (Life Science
Identifier) for the new species described here can be viewed through any standard web browser by appending the LSID to the prefix “http://zoobank.org/”. The LSID for this publication
is: urn:lsid:zoobank.org:pub:61380956-FlAC-46C0-84F3-ClED545C46DC.
Separate print-only edition of paper(s) (reprint) are available upon request as a print-on-demand service. Please inquire by sending a request to: Amphibian & Reptile Conservation
(amphibian-reptile-conservation.org; arc.publisher@gmail.com).
Amphibian & Reptile Conservation is a Content Partner with the Encyclopedia of Life (EOL); http:///www.eol.org/ and submits information about new species to the EOL freely.
Digital archiving of this paper are found at the following institutions: ZenScientist (http://www.zenscientist.com/index.php/filedrawer); Ernst Mayr Library, Museum of Comparative Zool-
ogy, Harvard University, Cambridge, Massachusetts (USA); Elorida Museum of Natural History, Gainesville, Elorida (USA).
Complete journal archiving is found at: ZenScientist (http://www.zenscientist.com/index.php/filedrawer); Florida Museum of Natural History, Gainesville, Florida (USA).
Citations
ICZN. 2012. Amendment of Articles 8,9,10,21 and 78 of the International Code of Zoological Nomenclature to expand and refine methods of publication. Zootaxa 3450: 1-7.
Polaszek A et al. 2005a. Commentary: A universal register for animal names. Nature 437 : 477.
Polaszek A et al. 2005b. ZooBank: The open-access register for zoological taxonomy: Technical Discussion Paper. Bulletin of Zoological Nomenclature 62(4): 210-220.
Amphib. Reptile Conserv. | amphibian-reptile-conservation.org (24)
May 2014 | Volume 8 | Number 1 | e76
Copyright: © 2014 Ron et al. This is an open-access article distributed under the terms
of the Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License,
which permits unrestricted use for non-commercial and education purposes only provided
the original author and source are credited. The official publication credit source: Amphib-
ian & Reptile Conservation at: amphibian-reptile-conservation.org
Reproduction and spawning behavior in the frog,
Engystomops pustulatus (Shreve 1941)
^Santiago R. Ron, ^’^Andrea E. Narvaez, and ^Giovanna E. Romero
^Museo de Zoologia, Escuela de Biologia, Pontificia Universidad Catolica del Ecuador, Av. 12 de Octubre y Roca, Aptdo. 17-01-2184, Quito,
ECUADOR ^La Trobe University, Department of Zoology, Bundoora VIC 3086, AUSTRALIA ^Museo Ecuatoriano de Ciencias Naturales, Herbario
Nacional del Ecuador, Av. Rio Coca E6-115 e Is la Fernandina, Quito, ECUADOR
Amphibian & Reptiie Conservation
[Special Section] 8(1): 25-32.
Abstract— The study of reproductive strategies is central to understand the demography of
populations and the energetic relationships of the species with their ecosystem. Documenting the
reproductive natural history of the species is pressing in groups, like amphibians, that are threatened
with extinction at a global scale. Herein, we describe the reproductive ecology and spawning
behavior of the leptodactylid frog Engystomops pustulatus. In addition, we report observations
that suggest the existence of an alternative mating strategy. Our results show that reproduction
in E. pustulatus is characterized by high maternal investment (15% egg mass relative to body
mass). We found evidence of size-assortative mating with a tendency of larger females to mate with
larger males. Clutch size was correlated with female weight, female condition and male size. Larger
females showed a tendency to lay larger foam nests and larger nests contained more eggs. At
reproductive choruses, there was a male-biased operational sex ratio, indicative of high variance in
male reproductive success. We observed an amplectant couple spawning while an additional male
was embedded in the foam. We hypothesize that this behavior is evidence of an alternative mating
strategy where a small non-amplectant male attempts to fertilize the eggs that are extruded by the
amplectant female.
Resumen. — El estudio de las estrategias reproductivas es fundamental para entender la demografia
de las poblaciones y las relaciones energeticas de las especies con su ecosistema. Documentar
la historia natural reproductiva de las especies es apremiante en grupos, como los anfibios, que
estan amenazados con extincion a nivel mondial. Aqui, describimos la ecologia reproductiva
y el comportamiento de anidacion en la rana leptodactilida Engystomops pustulatus. Ademas,
reportamos observaciones que sugieren la existencia de una estrategia reproductiva alterna.
Nuestros resultados indican que la reproduccion en E. pustulatus esta caracterizada por una alta
inversion energetics de la hembra (15% de masa de huevos en relacion a la masa corporal). Se
evidencia que el apareamiento es selective con respecto al tamaho, con una tendencia de hembras
grandes a aparearse con machos grandes. El tamaho de la puesta estuvo correlacionado con el peso
de la hembra, la condicion de hembra y el tamaho del macho. Las hembras mas grandes mostraron
una tendencia de poner nidos de espuma mas grandes y los nidos mas grandes tuvieron un mayor
numero de huevos. En cores reproductivos, hubo una tasa sexual operativa sesgada hacia los
machos, lo que indica una alta varianza en el exito reproductive de los machos. Se observe una
pareja en amplexus construyento un nido mientras un macho adicional estaba incrustado en el
nido de espuma. Hipotetizamos que este comportamiento evidencia una estrategia de apareamiento
alterna en la que un macho pequeho intents fertilizar huevos puestos por una hembra en amplexus
con otro macho.
Key words. Alternative mating strategy, clutch size, clutch piracy, fertilization rates, nesting behavior, testis size
Citation: Ron SR, Narvaez AE, Romero GE. 2014. Reproduction and spawning behavior in the frog, Engystomops pustulatus (Shreve 1941). Amphibian
& Reptile Conservation 8(1) [Special Section]; 25-32 (e79).
Correspondence. Emails: ^ santiago.r.ron@gmail.com (Corresponding author), ^aenarvaezg@gmail.com,
^giaromev@gmail. com
Amphib. Reptile Conserv. | amphibian-reptile-conservation.org (25)
August 2014 | Volume 8 | Number 1 | e79
Ron et al.
Introduction
Understanding the natural history of reproduction is
essential to characterize the ecological niche and the
survival prospects of amphibians. Acquiring a better
understanding of amphibian reproduction will assist con-
servation efforts in the vertebrate class with the higher
number of species threatened with extinction (Chanson
et al. 2008).
The deposition of eggs in foam nests characterizes
the reproduction of most species of the Neotropical fam-
ily Leptodactylidae, which has 201 species distributed
from southern Texas to southern Chile (Frost 2014). In
most species, males call in choruses that are visited by
receptive females, which then actively choose a mate
(Ryan 1985). Amplectant pairs build foam nests where
hundreds of eggs are laid and fertilized (Crump 1974;
Heyer 1969; Ryan 1985). The foam is formed when the
male kicks the jelly surrounding the eggs while the fe-
male discharges them. The foam may protect the eggs
from dehydration and/or predation (Duellman and Tmeb
1994; Menin and Giaretta 2003) or from excessive heat
(Gorzula 1977).
Foam nests may facilitate multiple paternity by retain-
ing sperm (Kusano et al. 1991). There is a high propor-
tion of foam-nesting species among known cases of mul-
timale spawning in anurans, eight out of 15 (Byrne and
Roberts 1999; Kaminsky 1997; Prado and Haddad 2003).
Although several reproductive characteristics of Lepto-
dactylidae should favor multiple male mating strategies,
there are only two documented cases, Leptodactylus
chaquensis and L. podicipinus (Prado and Haddad 2003).
The paucity of records may be partly due to lack of stud-
ies. Although several leptodactylid species are abundant
and live even in urban areas, little is known about its bi-
ology beyond brief accounts of its systematics and mor-
phology. Such is the case of the widely distributed and
abundant Engystomops pustulatus (Ron and Read 2012).
Herein, we describe the reproductive natural history
of Engystomops pustulatus including fertilization rates,
testis size, clutch size, and relative egg mass to explore
factors that influence mate choice and reproductive out-
put. We also describe its spawning behavior with obser-
vations suggest the existence of a secondary male mating
strategy.
Materials and Methods
Study site and species
Engystomops pustulatus inhabits dry shrub, deciduous
forest, and lowland moist forest below 300 m in western
Ecuador. It can be relatively common during the rainy
season, when they reproduce. They are explosive breed-
ers that congregate around temporal pools. Males call
from the water and amplectant pairs build foam nests to
deposit their eggs (Ron and Read 2012). Engystomops
Amphib. Reptile Conserv. | amphibian-reptile-conservation.org (26)
pustulatus should not be confused with E. pustulosus, a
Central American species that has been a model for stud-
ies of behavioral ecology (e.g., Ryan 2005). For clarity,
hereafter, we refer to E. pustulosus exclusively as “Tun-
gara frog.”
Operational sex ratio (e.g., the number of males rela-
tive to the number of females in breeding aggregations)
in Engystomops pustulatus was assessed in western
Ecuador at three localities: Reserva Cerro Blanco, (W
80.0214°, S 2.0264°, Provincia del Guayas; 19 March
2003), Patricia Pilar (Provincia Los Rios; 21 Eebmary
2002), and the town of La Mana (Provincia de Cotopaxi;
28 December 2003). Reproductive output, nest size and
size assortment were evaluated in La Mana (W 79.265°,
S 0.943°, elevation 160 m) between 28 December 2003
and 08 February 2004 and Patricia Pilar (W 79.3707°, S
0.5372°, elevation 200 m) between 23 January and 20
April 2008 during the rainy season. At La Mana and Pa-
tricia Pilar, the vegetation is Evergreen Lowland Moist
Forest (as deflned by Sierra et al. 1999). Most of the for-
est in the region has been converted to pastures and agri-
cultural lands. Field observations took place after dusk,
between 19:00 and 3:00 h. Breeding occurred in small
temporary ponds on the streets of the town. Some sites
were under dim artificial light (street poles).
Fertilization rates and nest size
We estimated fertilization rates from amplectant pairs
collected from the field. The amplectant pairs were placed
in individual circular plastic containers (10 cm diameter)
with water depth of one cm. Most pairs made a nest after
few hours. Three or four days later, we washed the foam
with a solution of chlorine and water and counted the
number of hatched and undeveloped eggs (as described
by Ryan 1983). We used this proportion as a proxy for
fertilization rates. This methodology does not allow dis-
criminating between undeveloped eggs as result of egg
unviability or failed fertilization. Therefore, our method-
ology may slightly underestimate fertilization rates.
To estimate nest size, we measured (with digital cali-
pers, to the nearest 0.01 mm) the length of the longest
axis, width at the widest point perpendicular to the lon-
gest axis, and height of all nests laid in the containers. We
estimated nest volume with the formula of Vi ellipsoid:
V = —abc
12
where a, b, and c are the length, width, and height, re-
spectively. The measurements were taken while the nests
were <1 day old.
Adult size and egg mass
Sex was determined by the presence of nuptial pads, vo-
cal sac folds, and/or by gonad inspection. Snout-vent
length (SVL) was measured with Fowler digital calipers
(nearest 0.01 mm). Body mass was measured in the field
August 2014 | Volume 8 | Number 1 | e79
Reproduction and spawning behavior in Engystomops pustulatus
(before and after oviposition in females) with a digital
balance (nearest 0.1 g). Relative egg mass (maternal in-
vestment) was calculated as 1 — the ratio (female mass
after oviposition/female mass before oviposition).
After being kept in the plastic containers to allow
spawning, females were euthanized by immersion in
chloretone, fixed in 10% formalin, and preserved in 70%
ethanol. Egg mass and body mass were measured after
preservation in females that did not spawn. Each female
was weighted on a digital balance (to the nearest 0.001
g), after removing excess ethanol. Then, the remaining
egg masses (including immature eggs and jelly) were
removed from the abdomen and weighted. Relative egg
mass was calculated by dividing total egg mass by non-
gravid female mass. Estimates of relative egg mass could
be influenced by preservation in ethanol. Therefore, com-
parisons with relative egg mass in non-preserved nesting
females should be interpreted with caution. All preserved
specimens are deposited at the amphibian collection of
the Zoology Museum of Pontificia Universidad Catolica
del Ecuador.
Reproductive behavior
Behavioral observations were carried out at male cho-
ruses in La Mana, Ecuador. Spawning behavior was
described from of a single nesting event at La Mana.
Spawning was recorded in the field under infrared light
with a digital camcorder SONY TRV70. The complete
video is available at AmphibiaWeb (http://amphibiaweb.
org).
Statistical analyses
Eor normally distributed variables, we tested the sig-
nificance of relationship between them using linear re-
gression ANOVAs; for non-normal variables, we tested
relationship with Spearman’s rank correlations. Differ-
ences between groups were tested with t-tests (assuming
non-equal variances). Statistical tests were implemented
in software IMP v.5.1 (SAS Institute, 2003).
Results
Reproductive output, fertilization rates, and
nest size
Among 77 nests, the mean number of eggs was 320 (SD
= 142.6, range 0-747). The average percentage of unfer-
tilized eggs was 1.89% (SD = 3.3, 0-19.1, n = 46); Pig.
lA); ~l/5 of the nests had a fertilization rate of 100%.
Snout- vent length difference between both parents was
not correlated with the number of unfertilized eggs
(Spearman’s Rho = 0.098, P = 0.524) or the proportion
of unfertilized eggs (Rho = 0.145, P = 0.341).
Mean nest volume was 37.0 cm3 (SD = 14.4, range
2.8-85.6, n = lA). Nest volume is correlated to the
number of eggs (larger nests have more eggs; Table
1, Pig. 2A) and female size (larger females lay larger
Amphib. Reptile Conserv. | amphibian-reptile-conservation.org (27)
-3 -2 -1 0 1 2 3 4 5 6
Pair SVL difference (mm)
25 26 27 28 29 30 31 32 33 34 35 36 37
SVL female (mm)
Fig. 1. Size and fecundity rates for amplectant pairs of Engysto-
mops pustulatus. After collected in amplexus in the field, pairs
were left in plastic containers where they could spawn. (A) Pro-
portion of unfertilized eggs among pairs that successfully built
a nest, (B) Female vs. male snout-vent length (SVL) with linear
regression and 95% confidence intervals (dashed lines).
nests; Table 1, Pig. 2B). A multiple regression of num-
ber of eggs, female SVL, and male SVL explains 25%
of the variation in nest volume {F = 7.51, df = 66, P
< 0.001). However, only number of eggs is signifi-
cant for the regression model (F = 18.18, P < 0.001).
Number of eggs was significantly correlated with
male SVL (Pig. 2C) but not with female SVL. Number
of eggs was correlated with female mass before and after
oviposition and female condition (Table 1).
Non-spawning females had large masses of eggs in
their abdomens (mean relative egg mass = 0.354, SD
= 0.138, range 0.129-0.621, n = 13). Average maternal
investment for spawning females was 15.2% of body
weight (SD = 7.77, 1.8-39.4, n = 42).
Size assortment and spawning
We found size-assortative mating as male and female
size of amplectant pairs was correlated (ANOVA’s F =
24.1, P < 0.001, R^ = 0.176; Pig. IB). Overall, females
were significantly larger than their mates (n= 115; mean
female SVL = 31.0 mm, SD = 1.9, range 25.3-36.5; male
August 2014 | Volume 8 | Number 1 | e79
Ron et al.
0 10 20 30 40 50 60 70 80 90
Nest volume (cm3)
Female SVL (mm)
Male SVL (mm)
Fig. 2. Bivariate plots for (A) nest volume vs. number of eggs,
(B) females size vs. nest volume, and (C) male size vs. num-
ber of eggs in Engystomops pustulatus. Linear regressions with
95% confidence intervals (dashed lines), determination coef-
ficients (R^), and ANOVA’s P values are shown.
SVL = 28.5, SD = 1.3, 25.2-32.3; paired-f = 14.7, df =
104, P < 0.001). However, in 10 pairs (8.6%) the male
was larger. Mean SVL difference between amplectant
male and female was 2.5 mm (SD = 1.74, range -2. 6-6.7,
n = 105).
Reproductive behavior
Males began calling immediately after dusk. They called
while floating in temporary ponds with water <10 cm
deep. Male density at some choruses was high, result-
ing in some males calling a few centimeters away from
Amphib. Reptile Conserv. | amphibian-reptile-conservation.org (28)
each other. Males defended calling sites and aggressive
interactions ensued if another male approached within
a radius of <10 cm. Aggressive behavior consisted of
mew-like vocalizations and attempts to clasp the rival
male. Amplexus and egg deposition occurred at the same
ponds where choruses were calling. Amplexus is axillary.
Operational sex ratio at choruses was male-biased.
During a survey at La Mana, we recorded 14 males but
only one female; at Cerro Blanco, the ratio was 3:1 (n =
16 individuals); at Patricia Pilar, the ratio was 8.5:1 (n =
19). The average ratio is 8.5:1 (n = 3 surveys).
Spawning behavior . — Nests were built while in am-
plexus, on shallow water, next to vegetation or muddy
banks. The following description is based on an amplec-
tant pair found on 04 February 2004 at -1:00 AM (male
QCAZ 26672, SVL = 26.2 mm, hereafter referred as
a-male; female QCAZ 26671, SVL = 31.7 mm) building
a foam nest. At the beginning of the observation, the nest
already had a diameter >50 mm. The male remained in
amplexus until the couple left the nest (50 minutes later).
To form the foam, the male kicked the egg mass while
they were being extruded from the female’s vent. Kick-
ing occurred in regular bursts with intervening periods
during which the couple was inunobile. In a typical burst
cycle, the male’s legs move downward, presumably to
place his feet next to the female vent. Then, the male’s
feet move up until they reach the posterior end of his
dorsum. At that moment, usually one or two eggs become
visible in the jelly matrix between the feet. This is fol-
lowed by a series of -20 rapid kicks on which his legs
become partly extended backward and then distended
forward until reaching the posterior end of his dorsum.
During these kicks, his legs move simultaneously but in
opposite directions (forward-backward) and feet momen-
tarily touch medially. The burst ends with to 2-A forceful
kicks on which his legs are nearly completely extended
posterolaterally, partly removing the foam that lies im-
mediately behind the couple. Each male burst seems to
be triggered by an abdominal movement of the female.
Each burst of kicking lasted on average 4.64 s (SD =
0.53, range 2.13-6.22, n = 215); the intervening inuno-
bile periods lasted 9.25 s (SD = 12.15, range 0.12-119,
n = 215). Total duration of bursts was 16’30” during 50’
of observation. The duration of each burst and the num-
ber of bursts decreased during the second half of the se-
quence (Fig. 3).
Multimale nesting behavior . — Multimale spawn-
ing was only observed once, during the spawning event
described in the previous section (male QCAZ 26672,
female QCAZ 26671). From the beginning of the obser-
vation, a peripheral adult male (QCAZ 26673; hereafter
referred as p-male) was sitting on the nest edge, directly
opposite to the nesting couple and with the posterior Vi of
its body embedded in the foam (Fig. 4). On at least flve
occasions its body moved slightly from side to side in se-
quences that lasted 3-4 s (Fig. 3). The movements were
always in concert with the kicking bursts of the a-male.
August 2014 | Volume 8 | Number 1 | e79
Reproduction and spawning behavior in Engystomops pustulatus
Table 1. Pearson’s correlation coefficients and ANOVA’s P values for linear regressions. Body condition is defined
as the residuals between SVL and mass. SVL = snout-vent length.
Variable 1
Variable 2
R2
n
P
Nest volume
Female size (SVL)
0.082
lA
0.013*
Nest volume
Female mass (before oviposition)
0.020
56
0.287
Nest volume
Male size (SVL)
0.026
lA
0.168
Nest volume
Male condition
<0.001
lA
0.796
Nest volume
Number of eggs
0.241
70
<0.001*
Number of eggs
Male size (SVL)
0.051
76
0.049*
Number of eggs
Female size (SVL)
0.027
76
0.151
Number of eggs
Female mass (before oviposition)
0.111
62
0.008*
Number of eggs
Female mass (after oviposition)
0.128
58
0.006*
Number of eggs
Female condition
0.125
62
0.005*
Number of eggs
Male condition
0.011
76
0.352
* Significant at P <0.05
Burst no.
10
^ 6
Z3
JD
N- 4
o ^
6
2 ! 2
0
Fig. 3. Spawning of Engystomops pustulatus nesting couple
(QCAZ 26671-72) and P-male (QCAZ 26673). Above: dura-
tion of kicking bursts. Below: number of bursts per minute; as-
terisks indicate P-male movements in the foam. Measurements
are shown in sequence from the beginning of the observation
until the couple left the nest. See text for details.
Most likely, the movements were generated by kicking
bursts of the p-male legs (hidden below the foam). He
left 23 min later, apparently following an amplectant
couple (not collected) that approached at a distance of 10
cm from the nest (see below).
The p-male (SVL = 25.3 mm) was one of the small-
est in the population. Out of 49 calling males measured
during the same season, only three were smaller (mean
SVL = 27.55 mm, SD = 1.23); out of 59 males found in
amplexus, only one was smaller (mean SVL = 27.9 mm,
SD = 1.20). Assuming a normal SVL distribution, the
probability of drawing a male with equal or lower SVL
by chance is 0.020 (z-score = -2.058). On a sample of
seven males including the p-male, mean testes mass was
0.47% of total body mass (range 0.24-0.70%; mean body
mass = 1.59 g, SD = 0.28). Contrary to our expectations,
the p-male had the proportionally smallest testes.
Discussion
Clutch size, fertilization success, and parental
investment
Number of eggs/clutch in Engystomops pustulatus is
-37% higher than in the tungara frog (Ryan 1985). In
several anurans, clutch size is significantly correlated to
body size (e.g.. Crump 1974; Ryan 1985; Wells 2007). In
E. pustulatus, such a relationship was significant for fe-
male condition and gravid and non-gravid female mass.
However, the relationship was not significant for female
SVL. Interestingly, we also found a significant correla-
tion between number of eggs and male SVL suggesting
that larger males have a higher reproductive success.
This correlation could not be explained by indirect cor-
relations with the other measured variables because they
were either uncorrelated with male SVL (e.g., nest vol-
ume) or uncorrelated with number of eggs (e.g., female
SVL).
Female SVL and number of eggs are correlated with
nest volume. Nest foam also results from intense male
physical activity. However, we were unable to find a re-
lationship between nest volume and either male size or
male body condition (Table 1).
We found size assortative mating as large females
have a tendency to mate with large males. An adaptive
explanation for size assortative mating states that it in-
creases fertilization rates because it results in female and
male vents being closer during amplexus (Licht 1976).
Evidence for this scenario has been reported for the Tun-
gara frog (Ryan 1985). Engystomops pustulatus lacks
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August 2014 | Volume 8 | Number 1 | e79
Ron et al.
Fig. 4. Engystomops pustulatus nesting couple (QCAZ 26671-
72) and P-male (QCAZ 26673). The couple builds the foam
nest as the male kicks the egg masses extruded by the female.
Frame from video (infra-red recording). See text for details.
Fig. 5. Relationship (in log space) for body and testis mass
among 11 species of Leptodactylinae frogs. Except for Engys-
tomops pustulatus, data is from Prado and Haddad (2003).
Open circles indicate species on which multimale spawning
has been reported. Note that E. pustulatus, in which multi-male
spawning apparently occurs, also has larger testis than other
Leptodactylinae.
that relationship as demonstrated by couples with large
differences in size {2-A mm) showing high fertilization
rates (Fig. lA). The lack of influence of size difference
on fertilization may be explained by our observation of
spawning behavior because the male uses his feet to drag
the eggs from the female’s underside to his own vent.
Therefore, the relative position of male and female vents
may have a minor influence in the relative position of
eggs and released sperm. Fertilization rates are generally
high (more than 98% on average) suggesting that size
differences between male and female have little influence
in individual fitness. Similar results have been reported
in other explosive breeding anurans like Lithobates syl-
vaticus (Howard and Kluge 1985) and Anaxyrus cogna-
tus (Krupa 1988).
Size assortative mating could also result from non-
adaptive interactions. If small males mating with large
females are more easily displaced than large males mat-
ing with large females, a size correlation will result
(e.g., Howard and Kluge 1985). This mechanism seems
unlikely in Engystomops. During our fieldwork with E.
pustulatus and with other species of Engystomops in the
Choco and the Amazon region, we never saw unmated
males attempting to displace amplectant males. Attempts
were rare in E. pustulosus and all of them were unsuc-
cessful (Ryan 1985). Therefore, an explanation for size
assortative mating in E. pustulatus and its sister species,
E. puyango (reported by Ron et al. 2010) is pending.
Reproductive investment (or effort) is a measure of
the allocation of energy in reproduction relative to total
energy (Pianka 2011). Theory predicts that a high repro-
ductive investment should be more adaptive if females
are unlikely to survive to another reproduction event
(Williams 1966). Our estimate of mean reproductive in-
vestment for Engystomops pustulatus (15.2%; egg mass
relative to body mass) is relatively high in comparison
to other anurans. For example. Crump (1974) and Prado
and Haddad (2005) report investments ranging from 3.1
to 18.2% for 34 Neotropical species (including nine lep-
todactylids). The investment of E. pustulatus, however,
is not the highest recorded for an anuran. For example,
the myobatrachid Crinia signifera invests 25.9% of the
gravid female mass in each spawning event (Lemckert
and Shine 1993). This high investment was interpreted as
resulting from a low probability of survival to additional
spawning events (Lemckert and Shine 1993). Similarly,
we hypothesize that the observed large investment in E.
pustulatus could result from low survival rates.
Nesting behavior
Overall, nest building behavior was similar to that re-
ported for the Tungara frog (Dalgetty and Kennedy
2010; Heyer and Rand 1977) and Physalaemus ephip-
pifer (Hodl 1990). The kicking bursts observed in E.
pustulatus are comparable to the “rotational movements”
described in P. ephippifer except that the legs seem to
extend further backwards in E. pustulatus (compared to
figure 5 in Hodl 1990).
Nest building is an energetically costly task (Ryan
1985) and the observed decrease in the frequency of
kicking bursts towards the end of spawning (Fig. 3) was
also reported in the tungara frog (Ryan 1985) and Phy-
salaemus ephippifer (Hodl 1990). As in Leptodactylus
labyrinthicus, the Tungara frog, and P. ephippifer, kick-
ing bursts seemed to be triggered by a female abdominal
movement (Heyer and Rand 1977; Hodl 1990; Silva et
al. 2005). The movie quality did not allow us to deter-
mine whether the decrease in burst frequency was male
or female-driven in E. pustulatus.
Multimale mating behavior
Our observation of more than one male spawning with a
female during oviposition suggests that multiple pater-
nity and alternative reproductive strategies may exist in
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August 2014 | Volume 8 | Number 1 | e79
Reproduction and spawning behavior in Engystomops pustulatus
Engystomops pustulatus. Although the P-male was not in
amplexus, its movements were similar and in synchrony
with those of the amplectant male, suggesting that it was
attempting to fertilize eggs (clutch piracy). A similar re-
productive behavior (with synchronic leg movements)
has been reported in Leptodactylus chaquensis although
with up to seven males in addition to the amplectant male
(Prado and Haddad 2003). Egg fertilization by periph-
eral non-amplectant males has also been demonstrated
in Chiromantis xerampelina, a foam-nesting rhacophorid
(Jennions and Passmore 1993).
The evolution of multimale spawning should be fa-
cilitated in reproductive systems where: (1) the opera-
tional sex ratio is strongly male biased, (2) fertilization is
external, (3) fecundity is high, and (4) eggs are spatially
aggregated (Byrne and Roberts 2004; Shuster and Wade
2003). All this characteristics are part of the reproduc-
tion of E. pustulatus. Therefore, the occurrence of multi-
male spawning was probable. As previously reported in
the Tungara frog (Ryan 1983), our data suggests that an
individual male is frequently unable to fertilize all the
eggs of a clutch, even in the absence of sperm competi-
tion. Although the presence of unfertilized eggs suggests
the potential for fitness gain of a p-male sneaking into
the nest of an amplectant pair, the proportion of unfertil-
ized eggs was typically low (1.89% on average). Higher
fitness gains for the p-male may result from sperm com-
petition.
We could not determine the frequency of multimale
spawning in the population. We observed monoandrous
spawning frequently and multimale spawning was only
recorded once, suggesting that it is relatively infrequent.
This is consistent with observations across a variety of
taxa showing that p-male strategies exist at a low fre-
quency in natural populations (Shuster and Wade 2003;
but see Byrne 2002; Jennions and Passmore 1993). The
low number of reports of multimale spawning among
leptodactylids is surprising because the characteristics of
the reproductive system of Leptodactylidae should favor
the evolution of secondary male mating strategies. The
paucity of known cases may be, at least partly, a sam-
pling artifact because the reproductive behavior has been
described in only few species.
Acknowledgments. — Fieldwork in 2003 and 2004
was funded by NSF IRCEB grant 0078150 to D. Can-
natella. Fieldwork in 2008 was funded by a DGA grant
from Pontificia Universidad Catolica del Ecuador to S.
R. Ron. The Ecuadorian Ministerio de Ambiente pro-
vided research and collection permits 004-IC-FAU-DPF,
and 006-IC-FAU-DBAP/MA. Fieldwork at la Mana was
assisted by F. P. Ayala, M. A. Guerra, and S. Padilla and
at Patricia Pilar by A. Argoti, P. Arias, F. Camacho, I.
Narvaez and A. Teran. Jose R. Ron, G. M. Melo, and
R. Valdivieso provided logistic support in Quito. Help-
ful comments for the manuscript were provided by X.
E. Bernal.
Amphib. Reptile Conserv. | amphibian-reptile-conservation.org (31)
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Received: 16 May 2014
Accepted: 22 July 2014
Published: 08 August 2014
Santiago R. Ron is the curator of amphibians and professor at the Pontificia Universidad Cat61ica del Ecuador
in Quito. His research focuses on the evolution and diversity of Neotropical amphibians and the evolution of
animal communication and sexual selection. In the area of conservation biology, Santiago is interested in the
study of amphibian extinctions in the Andes. He is a founding member of the Ecuadorian Academy of Sciences.
Andrea E. Narvaez is a Ph.D. student at La Trobe University (Australia) and holds a Master’s degree in
Integrative, Evolutionary Biology and Infectious Diseases from the Universite Eran9ois Rabelais de Tours
^ liW (Erance). She is currently investigating the evolution of visual signaling of Ecuadorian anoles. Her research
focus includes ecology and animal behavior, mainly to evaluate the evolution of communication systems,
sexual and natural selection. She is interested in the use of quantitative tools to measure animal behavior and
has experience working with a variety of animals (frogs, crickets, lizards).
Giovanna E. Romero is a Research Associate at Museo Ecuatoriano de Ciencias Naturales, Botany Section
(QCNE). She holds a bachelor degree in biological sciences from Pontificia Universidad Catblica del Ecuador
and a Master’s degree in Plant Biology from the University of Texas in Austin. She has worked for many years
in the Galapagos Islands and has a deep knowledge of their flora and fauna. She has been collaborating with
QCNE since 2012. She is interested in taxonomy and digital curation of ferns and lycophytes.
Amphib. Reptile Conserv. | amphibian-reptile-conservation.org (32)
August 2014 | Volume 8 | Number 1 | e79
Amphibian & Reptiie Conservation
8(1) [Special Section]: 33-44.
Copyright: © 2014 Guayasamin et al. This is an open-access article distributed under
the terms of the Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported
License, which permits unrestricted use for non-commercial and education purposes only
provided the original author and source are credited. The official publication credit source:
Amphibian & Reptile Conservation at: amphibian-reptile-conservation.org
High prevalence of Batrachochytrium dendrobatidis in an
Andean frog community (Reserva Las Gralarias, Ecuador)
^Juan M. Guayasamin, ^Mngela Maria Mendoza, "^Ana V. Longo,
"^Kelly R. Zamudio, and ^^Elisa Bonaccorso
^Centro de Investigacion de la Biodiversidad y Cambio Climdtico (BioCamb), Universidad Tecnologica Indoamerica, Calle Machala y Sabanilla,
Quito, ECUADOR ^Laboratorio de Macroecologia, Centro de Investigaciones en Ecosistemas, Universidad Nacional Autonoma de MEXICO ^Grupo
de Investigacion en Ecologia y Conservacion Neotropical, SAMANEA Eundacion de Apoyo Educative e Investigative, COLOMBIA "^Department of
Ecology and Evolutionary Biology, Cornell University, Ithaca, New York, USA ^Biodiversity Institute, University of Kansas, Lawrence, Kansas, USA
Abstract— Wfe report patterns of infection of Batrachochytrium dendrobatidis {Bd) in a cloud forest
amphibian community in the Andean Western Cordillera of Ecuador (Reserva Las Gralarias). Data
were obtained during the rainy seasons of two consecutive years, using qPCR (year 2012) and end-
point PCR (year 2013). We show that average Bd prevalence in this amphibian community is high
(2012: 35-49%; 2013: 14-32%), but found no evidence of population declines or that Ed is negatively
affecting host populations. We found a significant correlation between Ed prevalence and taxonomy,
reproductive mode, and habitat, but no correlation between Ed infection intensity and the same three
variables. Contrary to our expectations, frog species with aquatic reproductive modes (glassfrogs,
treefrogs) showed lower Ed prevalence than direct-developing frogs {Pristimantis spp.). Although
further monitoring is needed to determine long-term population trends, our two-year dataset on
disease and population size support the hypothesis that frogs are tolerant to infection, a condition
that could potentially have resulted from exposure to previous Ed epidemic outbreaks.
Resumen. — En este estudio reportamos dates sobre los patrones de infeccion de Batrachochytrium
dendrobatidis {Bd) en una comunidad de anfibios en la Cordillera Occidental de los Andes del
Ecuador (Reserva Las Gralarias). Los dates fueron obtenidos durante la estacion Iluviosa en dos
ahos consecutivos, utilizando qPCR (aho 2012) y PCR de punto final (aho 2013). Los resultados
muestran una alta prevalencia de Eden la comunidad (2012: 35-49%; 2013: 14-32%); sin embargo, no
se encontro evidencia de disminuciones poblacionales o de que Ed este afectando negativamente a
las especies de anfibios. Existe una relacion significativa entre la prevalencia de Edy la taxonomia,
modo reproductive y habitat de los anfibios, pero no hubo correlacion entre la intensidad de
infeccion de Ed y las mismas tres variables. Contrario a nuestras predicciones, las especies de
anuros con larvas acuaticas (ranas de cristal, ranas arboreas) presentaron prevalencias de Ed mas
bajas que los anuros de desarrollo directo {Pristimantis spp.). A pesar de que se requiere de un
monitoreo continue para determinar las dinamicas poblacionales a largo plazo, los dates obtenidos
hasta el memento apoyan un escenario donde las especies de ranas de la Reserva Las Gralarias
parecen tolerar la infeccion de Ed, una condicion posiblemente adquirida mediante la exposicion a
brotes epidemicos previos.
Key words. Chytridiomycosis, emerging disease, amphibian declines, Andes, conservation
Palabras claves. Quitridiomicosis, enfermedad emergente, declinacion de anfibios, Andes, conservacion
Citation: Guayasamin JM, Mendoza AM, Longo AV, Zamudio KR, Bonaccorso E. 2014. High prevalence of Batrachochytrium dendrobatidis in an Andean
frog community (Reserva Las Gralarias, Ecuador). Amphibian & Reptiie Conservation 8(1) [Special Section]: 33-44 (e81).
Correspondence. Emails: ^jmguayasamin® gmail.com (Corresponding author).
August 2014 | Volume 8 | Number 1 | e81
Amphib. Reptile Conserv. | amphibian-reptile-conservation.org (33)
Guayasamin et al.
Introduction
A third of global amphibian species are threatened with
extinction (Stuart et al. 2004; Wake and Vredenburg
2008) and, most concerning, numerous local popula-
tion declines and extinctions have occurred in relatively
pristine areas, where anthropogenic habitat destruction is
low (Lips 1998, 1999; Drost and Fellers 1996; La Marca
et al. 2005). In the last two decades, several studies have
attributed anuran mass mortality events to the emergence
of the pathogenic fungus Batrachochytrium dendroba-
tidis (Bd), a pathogen with widespread geographic and
ecological distribution (Berger et al. 1998; Daszak et al.
1999, 2003; Lips et al. 2006; Becker and Zamudio 2011;
Rodriguez et al. 2014). Alternative explanations to am-
phibian declines add a role to global warming and tem-
perature variability (Pounds et al. 2006; Rohr and Raff el
2010; Menendez-Guerrero and Graham 2013).
Batrachochytrium dendrobatidis infects the keratin-
ized skin of amphibians and disrupts the regulatory func-
tioning of the integument (Berger et al. 1998; Voyles et
al. 2009). Infection inhibits host inunune responses in
some species (Fites et al. 2013) and in severe cases of
infection, electrolyte depletion and osmotic imbalance
may lead to mortality (Voyles et al. 2007, 2009). How-
ever, not all amphibian species are equally susceptible to
the pathogen. For example, at Santa Fe, Panama, Bd has
caused declines or local extinctions of most anurans in
the original community, but six species of frogs and toads
remain abundant, despite being infected by the fungus
(Lips et al. 2006). In laboratory challenge experiments,
amphibian mortality rates range from 0% to 100%, de-
pending on the species, host age, pathogen genotype, and
dosage (Berger et al. 2005a; Daszak et al. 2004; Longo et
al. 2014). The reasons for host differences in susceptibil-
ity include immunogenic variation (Ellison et al. 2014;
Savage et al. 2014), microhabitat use (Kriger and Hero
2007; Griindler et al. 2012), association with water as
embryos, tadpoles, or adults (Lips et al. 2003), and host
thermoregulatory behavior (Richards-Zawacki 2010).
Because Bd transmission may happen through frog-frog
contact, or through motile zoospore movement from one
host to another, frogs and toads that spend more time in
water are expected to have higher exposure and suscep-
tibility to infection than species that are primarily terres-
trial (i.e, direct developers; Lips et al. 2003; Kriger and
Hero 2007).
In this study, we report data on infection patterns of
Bd obtained during the rainy seasons of two consecu-
tive years in the amphibian community of Reserva Las
Gralarias, a cloud forest site in the Andean Western Cor-
dillera of Ecuador. We found that Bd prevalence in all
amphibian species is high, but found no evidence that
Bd is negatively affecting amphibians (i.e., no apparent
population declines, or records of clinical signs of chy-
tridiomycosis). We report on infection prevalence and
intensity dynamics for the two-year period, and examine
Amphib. Reptile Conserv. | amphibian-reptile-conservation.org (34)
patterns of Bd infection in species that vary in their tax-
onomy, reproductive mode, and habitat.
Materials and Methods
Study site: The study was conducted at Reserva Las
Gralarias (0°0L S, 78°44’ W; 1822-2400 m), a private
reserve covering an area of 1,063 acres (425 ha) located
on the Pacific slopes of the Andes, Pichincha Province,
Mindo Parish, Ecuador. The study site has an eleva-
tional range of 1,825-2,400 m and includes primary and
secondary forest, regenerating pasture, and numerous
ephemeral and permanent streams and creeks (Hutter and
Guayasanfin 2012).
Amphibian taxonomy: For generic and suprageneric clas-
sification, we follow the taxonomic proposals of Hedges
et al. (2008), Guayasamin et al. (2009), and Faivovich et
al. (2005), Pyron and Wiens (2011), as sununarized in
Frost (2014).
Amphibian richness and abundance: During the rainy
seasons of 2012 (23 January-29 March) and 2013 (14
March-22 April), we sampled trails of Reserva Las
Gralarias during the night, including most of its habitat
heterogeneity and elevational gradient, to record the spe-
cies richness of the reserve. We placed eight transects,
each with an area of 500 x 4 m (Appendix 1), to maxi-
mize species detection and to obtain a baseline dataset
on population size and Bd prevalence. Each transect was
sampled by two people for 3-4 hours during the night
(generally starting at 8 pm); temperatures during sam-
pling varied between 11-15 °C. All detected amphibians
were, when possible, photographed. Calling males were
also reported and identified with the aid of photographic
and acoustic guides (Arteaga et al. 2013; Centro Jambatu
2011-2014). We used a Student’s t-test to quantify differ-
ences in population sizes in transects that were sampled
multiple times during the rainy season of 2012 and 2013
(Lucy’s Creek and Kathy’s Creek); the normality of spe-
cies abundance was assessed using a Shapiro-Wilk Test.
Diagnosis of Batrachochytrium dendrobatidis: We
swabbed the ventral regions of all amphibians captured
in our survey, following the standard procedures in Hyatt
et al. (2007; Fig. 1); dry swabs were stored in -4 °C until
analysis. Testing for Bd was carried out using Real-Time
PGR (q-PCR) for samples obtained during 2012 and end-
point Polymerase Chain Reaction (PCR) for samples ob-
tained in 2013; the use of these two methods was contin-
gent on access to q-PCR (available during 2012). In both
cases, DNA extractions were carried out using guanidin-
ium thiocyanate. For samples obtained during 2012, we
used a 1:10 dilution of the extract as template in Taqman
q-PCR assays for the detection of Bd (Boyle et al. 2004).
This assay uses BJ-specific primers ITS 1-3 Chytr and
5.8S Chytr, in addition to the fluorescently-labeled probe
Ch)4r MGB2, and amplifies the ITS-1 fragment of the
August 2014 | Volume 8 | Number 1 | e81
Batrachochytrium dendrobatidis in an Andean frog community
Fig. 1. Swab sample obtained from Centrolene heloderma at
Reserva Las Gralarias, Ecuador.
Bd genome at the junction of the ITS-1 and 5.8S regions.
We used a standard curve that included 1000, 100, 10,
1, and 0.1 zoospore genome equivalents, and followed
qPCR conditions described in Boyle et al. (2004). For
samples obtained during 2013, Bd presence was tested
using the internal transcribed spacer regions (ITS-1, ITS-
2) primers Bdla (5’-CAGTGTGCCATATGTCACG-3’)
and Bd2a (5’-CATGGTTCATATCTGTCCAG-3’) de-
veloped by Annis et al. (2004); the presence/absence of
Bd was determined via the visualization of the amplified
band in agarose gel electrophoresis. The two methods to
detect Bd have different sensitivities; therefore, direct
comparisons of Bd prevalence between years should be
considered with caution. However, family and habitat
correlates with infection status should not be biased by
detection method, and qPCR offers the additional advan-
tage of quantifying infection intensity (load).
Prevalence and correlates of Batrachochytrium den-
drobatidis in amphibians: We estimated prevalence of
Bd within each anuran species as the number of frogs
that tested positive for Bd, divided by the total number of
sampled frogs for that particular species in a given year.
We estimated the 95% confidence interval for preva-
lence in each species, (Wilson 1927; Newcomb 1998).
We modeled Bd presence or absence in each individual
by using a logistic regression. We tested for possible as-
sociations of Bd prevalence with the following variables:
habitat (terrestrial, riparian, lentic), reproductive mode
(aquatic, terrestrial), and taxonomy (family). Statistical
significance of results was assessed with a chi-square
test.
Infection intensity of Batrachochytrium dendrobati-
dis and correlates in amphibians: We tested for possible
associations of Bd intensity (measured as zoospore ge-
nomic equivalents) with the following variables: habitat
(terrestrial, riparian, lentic), reproductive mode (aquatic,
terrestrial), and taxonomy (family, genus, species; Ap-
pendix 2). Given the strong right skew of infection load,
we used the non-parametric Kruskal- Wallis test. All sta-
tistical analyses were performed using R v. 2.15.3 (R
CoreTeam 2012).
Results
Species richness and abundance: During the two sam-
pling periods, we recorded a total of 2,450 individuals
of 28 species (Appendix 2). The abundance of species at
Lucy’s Creek and Kathy’s Creek is summarized in Tables
1 and 2. Because most taxa were scarce, we restricted
the comparisons between years to relatively abundant
species (glassfrogs). Abundances of glassfrogs at Lucy’s
Creek and Kathy’s Creek were not significantly different
between years (Tables 1, 2).
Prevalence of Batrachochytrium dendrobatidis in
amphibians: Swabs of 320 frogs were tested for Bd, and
O
Fig. 2. Significant Bd infection differences in amphibians according to reproductive modes, habitat use, and taxonomy. P values are
reported for 2012 and 2013; significance is noted by ** (p < 0.01) and *** (p < 0.001).
August 2014 | Volume 8 | Number 1 | e81
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Guayasamin et al.
Table 1. Abundance of amphibian species at Lucy’s Creek, Reserva Las Gralarias. Abundances are presented as minimum-maxi-
mum, followed, in parenthesis, by mean + standard error. The Student’s t-test was performed only in species with normally distrib-
uted abundances.
Lucy’s Creek
Mest (p)
Population trend
Year
2012
2013
Number of nights sampled
10
6
Family/Species
Centrolenidae
Centrolene lynchi
5 - 20 ( 13 . 1 + 5 . 13 )
2-18 ( 9.0 + 6 . 54 )
0.184
No difference
Centrolene peristictum
4-35 ( 20.7 + 11 . 68 )
6 - 26 ( 15.2 + 7 . 00 )
0.314
No difference
Nymphargus grandisonae
Hylidae
1 - 10 ( 6.2 + 3 . 12 )
0-8 ( 3.8 + 2 . 93 )
0.155
No difference
Hyloscirtus alytolylax
Craugastoridae
3-7 ( 4.3 + 1 . 42 )
0 - 6 ( 3.3 + 2 . 16 )
0.295
No difference
Pristimantis achatinus
0-2 ( 0.3 + 0 . 67 )
0-1 ( 0.2 + 0 . 41 )
—
—
Pristimantis appendiculatus
0-2 ( 0.8 + 0 . 92 )
0-1 ( 0.5 + 0 . 55 )
—
—
Pristimantis calcarulatus
0-2 ( 0.2 + 0 . 63 )
0-1 ( 0.2 + 0 . 41 )
—
—
Pristimantis eremitus
0
0-2 ( 0.5 + 0 . 84 )
—
—
Pristimantis eugeniae
0
0-2 ( 0.3 + 0 . 82 )
—
—
Pristimantis illotus
0-1 ( 0 . 1 + 0 . 32 )
0
—
—
Pristimantis parvillus
0-1 ( 0 . 1 + 0 . 32 )
0
—
—
Pristimantis sobetes
0
0-2 ( 0.3 + 0 . 82 )
—
—
Pristimantis w-nigrum
0-2 ( 0.6 + 0 . 84 )
0-2 ( 0.7 + 0 . 82 )
—
—
approximately a third of those were positive. In samples
from 2012, prevalence of Bd was relatively high, with
42% of all frogs testing positive for Bd infection. Dur-
ing 2013, Bd prevalence was 22%. Differences in preva-
lence between the two years are likely caused by detec-
tion method. Most species infected in 2012 carried low
Bd loads as determined by qPCR; the highest Bd load
obtained was in Centrolene ballux with 22.5 genomic
equivalents. Prevalence per species per year is summa-
rized in Table 3.
The logistic regression shows a significant relation-
ship (p < 0.001) of Bd infection with species reproduc-
tive mode, habitat, and taxonomy (Fig. 2). Frogs with
a terrestrial reproductive mode (direct developers; i.e.,
genus Pristimantis; see Duellman and Trueb 1986) have
a higher Bd prevalence than amphibians with aquatic re-
production (i.e., glassfrogs and treefrogs). Frog species
that are dependent on riverine habitats for reproduction
show significantly less infection than anurans that use
terrestrial or lentic habitats for reproduction (p < 0.001).
Also, species in the Centrolenidae family (glassfrogs)
show a lower Bd prevalence than species in Craugastori-
dae and Hylidae (Table 3). Although, Bd prevalence dur-
ing 2012 was significantly higher than in 2013 (probably
as a result of higher sensitivity of qPCR), we found no
significant interaction among sampling year and repro-
ductive mode, habitat, or taxonomy.
Infection intensity of Batrachochytrium dendrobati-
dis and correlates in amphibians: We found no relation-
ship between Bd infection intensity {Bd load, year 2012)
and taxonomy, reproductive mode, or habitat.
Discussion
Our results show a relatively high mean prevalence of
Bd (36%) across both years in the Andean frog concunu-
nity of Reserva Las Gralarias (see Hossack et al. 2010 for
comparison). From a total of 20 species analyzed, only
three (Nymphargus griffithsi, Pristimantis illotus, and P.
pteridophilus) tested negative for Bd\ however, sample
sizes for non-infected species were low (5, 1, and 3 in-
dividuals, respectively). Infected species included frogs
with very different reproductive modes, including taxa
with terrestrial direct development {Pristimantis spp.),
species that deposit eggs in ponds {Dendropsophus car-
nifex), and others that place their eggs on vegetation from
where hatching tadpoles drop into streams {Centrolene
spp., Nymphargus spp., Hyloscirtus spp.).
Because Bd is an aquatic pathogen (Berger et al.
2005b) we expected amphibian species with aquatic re-
productive modes to show higher infection prevalence
(Lips et al. 2005; Brem and Lips 2008). In fact, the most
dramatic amphibian declines and extinctions in the Andes
have occurred in species with aquatic larvae (La Marca
et al. 2005; Bustamante et al. 2005; Merino-Viteri et al.
2005; Coloma et al. 2010). Our results indicate, surpris-
ingly, a higher Bd prevalence in frogs with a terrestrial
reproductive mode {Pristimantis spp.) than in those that
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Batrachochytrium dendrobatidis in an Andean frog community
Table 2. Abundance of amphibian species at Kathy’s Creek, Reserva Las Gralarias. Abundances are presented as minimum-maxi-
mum, followed, in parenthesis, by mean + standard error. The Student’s t-test was performed only in species with normally distrib-
uted abundances.
Kathy’s Creek
f-test
Population trend
Year
2012
2013
Number of nights sampled
10
5
Family/Species
Centrolenidae
Centrolene ballux
5-37 ( 22.7 + 11 . 6 )
3-25 ( 11.4 + 8 . 67 )
0.078
No difference
Centrolene peristictum
0-5 ( 2.1 + 1 . 66 )
0-5 ( 2 . 0 + 1 . 87 )
0.918
No difference
Nymphargus grandisonae
0 - 7 ( 3.7 + 2 . 21 )
0-6 ( 2.2 + 2 . 28 )
0.242
No difference
Nymphargus griffithsi
0-8 ( 2.3 + 2 . 26 )
0-3 ( 1 . 4 + 1 . 34 )
—
—
Nymphargus lasgralarias
Hylidae
3-28 ( 19.4 + 8 . 54 )
7-28 ( 15.0 + 8 . 69 )
0.366
No difference
Hyloscirtus alytolylax
Craugastoridae
0-1 ( 0 . 1 + 0 . 32 )
0-1 ( 0.4 + 0 . 59 )
—
—
Pristimantis achatinus
0-3 ( 0.3 + 0 . 95 )
0-1 ( 0.2 + 0 . 45 )
—
—
Pristimantis appendiculatus
0 - 7 ( 1.2 + 2 . 10 )
0-1 ( 0.4 + 0 . 59 )
—
—
Pristimantis calcarulatus
0-3 ( 1 . 1 + 0 . 74 )
0-3 ( 1 . 0 + 1 . 23 )
—
—
Pristimantis eremitus
0-1 ( 0 . 1 + 0 . 32 )
0-1 ( 0.2 + 0 . 45 )
—
—
Pristimantis eugeniae
0-1 ( 0 . 1 + 0 . 32 )
1-2 ( 0.8 + 0 . 84 )
—
—
Pristimantis sobetes
0-1 ( 0.2 + 0 . 42 )
0-2 ( 0.4 + 0 . 89 )
—
—
Pristimantis w-nigrum
0-1 ( 0 . 1 + 0 . 32 )
0-1 ( 0.4 + 0 . 59 )
—
—
reproduce in water (mainly glassfrogs; Centrolene spp.,
Nymphargus spp.). This finding supports the idea that
even terrestrial breeders may serve as reservoirs for the
pathogen in diverse amphibian communities (Longo et
al. 2013). Higher prevalence in terrestrial frogs requires
that Bd zoospores survive in terrestrial habitats. John-
son and Speare (2003) indicated that Bd can survive in
moist soil for up to three months. Cloud forests in west-
ern Ecuador typically have near constant rain and high
levels of humidity during the rainy season (Hutter and
Guayasamin 2012; Arteaga et al. 2013), and this may ex-
tend zoospore survival in terrestrial environments at Las
Gralarias. Higher Bd prevalence in terrestrial frogs com-
pared to that in frogs with aquatic reproduction might
also be related to intrinsic differences in, for example, the
efficacy of immune responses (Rosenblum et al. 2009;
Woodhams et al. 2007) or differences in anuran skin mi-
crobiota (Flechas et al. 2012).
Our results also show that Bd prevalence is signifi-
cantly associated with taxonomy (i.e., family). Thus,
glassfrogs (family Centrolenidae) might have innnune
responses or skin microbiota that work as better barri-
ers to the pathogen than those in terrestrial (i.e., Pristi-
mantis) frogs. The strong correlation of prevalence with
taxonomy, habitat, and reproductive mode (Appendix 2)
indicates that further studies need to focus on the specific
effects of each of these factors; in other words, phytog-
eny (and taxonomy) correlates with reproductive mode
and habitat use.
A second surprising finding of our study is that, al-
though prevalence of Bd is high in most anuran species,
we did not observe any sign of population declines or
abrupt crashes, nor have we found dead or sick frogs dur-
ing four years of intensive fieldwork (2010-2014; JMG
pers. obs.). Thus, this frog connnunity persists with an
endemic pathogen and with relatively low loads (less
than 10 zoospores. Table 3). The apparent increased re-
sistance or tolerance of amphibians from Reserva Las
Gralarias to Bd infection may be explained by one or
several of the following mechanisms: (i) amphibian in-
nate and/or acquired defense mechanisms (Savage and
Zamudio 2012; Woodhams et al. 2007); (ii) skin bacte-
rial connnensals with anti-fungal properties (Harris et al.
2006); (iii) behavioral and ecological factors that reduce
the likelihood of infection and disease (e.g., microhabitat
selection, reproductive mode; Lips et al. 2003; Rowley
and Alford, 2007), and/or (iv) variation in Bd virulence
(Berger et al. 2005a; Fisher et al. 2009). Our amphib-
ian monitoring took place during the rainy season when
most species are active. Therefore, future studies should
determine whether or not this apparent tolerance to Bd
is stable through longer periods of time or if it fluctuates
depending on environmental variables influencing host
innnunity, behavior, microbiota, or pathogenicity.
After the emergence of an infectious disease, surviv-
ing hosts can evolve tolerance or resistance (Retallick et
al. 2004; Savage and Zamudio 2011). The earliest known
record of Bd in Ecuador is in 1980, in the Harlequin frog
Atelopus bomolochos (Ron and Merino-Viteri 2000),
a species that is now probably extinct (Coloma et al.
2014). If Bd reached and spread in Ecuador during the
early 1980s (Ron et al. 2003; Lips et al. 2008), we hy-
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Guayasamin et al.
Table 3. Prevalence of Batrachochytrium dendrobatidis (Bd) in amphibians at Reserva Las Gralarias, Ecuador, during the rainy
seasons of 2012 and 2013. Bd prevalence for each species is followed, in parenthesis, by a 95% confidence interval. Bd load sum-
mary data includes only samples that tested positive for Bd.
2012
2013
N
Positive Bd
Prevaience
Bd Load
(mean ± sd)
N
Positive Bd
Prevaience
Family: Centrolenidae
Centrolene ballux
17
8
47%
(24-71%)
6.5 + 10.7
9
2
22%
(39-59%)
Centrolene heloderma
6
1
17%
(1-63%)
0.6
1
0
0%
(0-95%)
Centrolene lynchi
6
1
17%
(1-63%)
-
5
1
20%
(1-70%)
Centrolene peristictum
21
6
29%
(12-52%)
2.1 + 1.5
16
3
19%
(5-46%)
Nymphargus grandisonae
21
5
24%
(9-48%)
4.7
-
-
-
Nymphargus griffithsi
3
0
0%
(1-69%)
-
2
0
0%
(0-80%)
Nymphargus lasgralarias
16
6
38%
(16-64%)
6.5 + 1.5
10
2
20%
(4-56%)
Family: Hylidae
Dendropsophus carnifex
10
5
50%
(20-80%)
-
-
-
-
Hyloscirtus alytolylax
9
8
89%
(51-99%)
2.4 + 2.4
7
2
29%
(5-70%)
Family: Craugastoridae
Pristimantis achatinus
7
4
57%
(20-88%)
-
-
-
-
Pristimantis appendiculatus
23
10
44%
(24-65%)
1.9 + 2.4
-
-
-
Pristimantis calcarulatus
15
2
13%
(2-42%)
1.1
15
1
7%
(4-34%)
Pristimantis eremitus
4
4
100%
(40-100%)
0.9 + 0.2
4
2
50%
(9-91%)
Pristimantis eugeniae
18
12
66%
(41-86%)
2.5
2
1
50%
(3-97%)
Pristimantis hectus
8
2
25%
(4-64%)
-
14
4
29%
(10-58%)
Pristimantis illotus
-
-
-
-
1
0
0%
(0-95%)
Pristimantis parvillus
9
4
44%
(15-77%)
-
-
-
-
Pristimantis sobetes
8
3
38%
(10-74%)
-
9
3
33%
(9-69%)
Pristimantis pteridophilus
-
-
-
-
3
0
0%
(0-69%)
Pristimantis w-nigrum
21
13
62%
(39-81%)
1.5 + 0.7
-
-
-
TOTAL
222
94
42%
(35-49%)
88
19
22%
(14-32%)
pothesize that many of the population declines observed
in the country at that time (e.g., Coloma 1995, 2002;
Coloma et al. 2000; Ron et al. 2003; Bustamante et al.
2005; La Marca et al. 2005; Lips et al. 2008; Coloma et
al. 2010) could be attributable to chytridiomycosis. Thus,
it is probable that most Andean amphibian communities
have been exposed to Bd for more than three decades and
that current sampling finds remnant species that are tol-
erant to Bd while the susceptible species are already ex-
tinct. Under this scenario, selection should have favored
the persistence of amphibian species or specific popula-
tions that have developed defenses against Bd; therefore.
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Amphib. Reptile Conserv. | amphibian-reptile-conservation.org (38)
Batrachochytrium dendrobatidis in an Andean frog community
changing host composition of these communities right
after pathogen emergence. We acknowledge, however,
that this is a working hypothesis which assumptions
depend on our knowledge of the historical distribution
of the chytrid. For example, if new data shows that Bd
was present in the Ecuadorian Andes before amphibian
declines were noticed, such piece of information would
support the endemic pathogen hypothesis, which states
that environmental changes triggered Bd outbreaks (Ra-
chowicz et al. 2005).
Reserva Las Gralarias is one of the most studied and
species-rich area in the cloud forest of the tropical An-
des, containing numerous species considered endangered
by the lUCN (2014; see Appendix 2). However, the
community (and surrounding areas) lacks at least three
groups of species that were conspicuous in Ecuadorian
cloud forests: marsupial frogs {Gastrotheca plumbea,
G. guentheri), harlequin frogs {Atelopus longirostris, A.
mindoensis), and dendrobatid frogs (Hyloxalus lehmani,
H. maquipucuna) (Coloma et al. 2011-2014; Arteaga et
et al. al. 2013). Marsupial and harlequin frogs are par-
ticularly susceptible to Bd (Lips et al. 2003; Elechas et al.
2012; Ellison et al. 2014; DiRenzo et al. 2014) and are
the primary species that suffered population declines and
extinctions in Ecuador (Lips et al. 2002; La Marca 2005)
even in pristine areas. The absence of these lineages at
Reserva Las Gralarias supports to the hypothesis that this
is a post-decline amphibian community. Understanding
the long-term effects of pathogens (eg., chytrid) and tem-
perature variability in such a community is essential for
the continued effective management of endangered spe-
cies in the Andean cloud forests.
Considering Ecuador’s high diversity of amphibian
species, life history modes, and evolutionary history, our
study provides a baseline to study the evolution of de-
fense strategies against Bd. We reconnnend further re-
search to determine the mechanisms driving the observed
differences in pathogen exposure among hosts differing
in reproductive modes, habitat, and taxonomy.
Acknowledgments. — Previous versions of this article
greatly benefited from the reviews of Luis A. Coloma,
and five anonymous reviewers. This study was funded
by the lUCN Save-Our-Species (SOS) program and Uni-
versidad Tecnologica Indoamerica, through the project
“Conservation of Endangered Species in the Choco Bio-
geographic Zone: Integrating habitat management, bio-
logical monitoring, and community outreach.” SOS is a
joint initiative of lUCN, the Global Environment Eacility
and the World Bank; its objective is to ensure the long-
term survival of threatened species and their habitats.
AMM’s research was supported by a scholarship from
the program “Becas Mixtas del Consejo Nacional de
Ciencia y Tecnologfa, CONACYT,” Mexico. Idea Wild
kindly granted equipment for AMM’s field work. Reser-
va Las Gralarias (Jane A. Lyons) provided invaluable lo-
gistic support throughout the project. Special thanks to
Amphib. Reptile Conserv. | amphibian-reptile-conservation.org (39)
Henry Imba, Italo G. Tapia, Lucas Bustamante, and Jai-
me Garcia for assistance during fieldwork. JMG thanks
AA for the one-day delay in Miami, where the final ver-
sion of the ms was finished. Diana Flores provided as-
sistance with molecular diagnosis of Bd. Special thanks
to John Kelly for his help in statistical analyses. Research
permits were issued by the Ministerio de Ambiente, No
05-2013-IC-FAU-DPAP-MA.
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Received: 19 June 2014
Accepted: 04 August 2014
Published: 28 August 2014
Appendix 1. Transects sampled at Reserva Las Gralarias. Each transect has an area of 500 x 4 m. Latitude and longitude are in
decimal degrees.
Transect
Elevation (m)
Latitude
Longitude
Habitat
Lucy’s creek
1822-1858
At start: -0.00492
At end: -0.00342
At start: -78.73344
At end: -78.74051
Riverine vegetation along creek
Kathy’s creek
2041-2066
At start: -0.01696
At end: -0.0156
At start: -78.7314
At end: -78.73386
Riverine vegetation along creek
Santa Rosa river
1884-1882
At start: -0.0133
At end: -0.01054
At start: -78.72368
At end: -78.7211
Riverine vegetation along river
Waterfall trail
1897-2107
At start: -0.0135
At end: -0.01379
At start: -78.72461
At end: -78.7269
Primary and secondary terra firme forest
Five-Frog creek
2141-2156
At start: -0.03166
At end: -0.03098
At start: -78.70421
At end: -78.70853
Riverine vegetation along creek
Osoverde & Guarumo trail
2141-2156
At start: -0.03166
At end: -0.03098
At start: -78.70421
At end: -78.70853
Primary and secondary terra firme forest
Puma trail
1923-2031
At start: -0.00954
At end: -0.00708
At start: -78.7346
At end: -78.73662
Primary and secondary terra firme forest
TKA trail
2192-2216
At start: -0.0275
At end: -0.02516
At start: -78.70477
At end: -78.70353
Primary and secondary terra firme forest
Peccary trail
1803-1896
At start: -0.00750
At end: -0.0076
At start: -78.72635
At end: -78.72862
Primary and secondary terra firme forest
Appendix 2. Amphibians at Reserva Las Gralarias, with corresponding lUCN (2014) conservation status. The list includes three
potential new species (Pristimantis sp. 1, Pristimantis sp. 2, and Pristimantis sp. 3). Reproductive modes are sensu Haddad and
Prado (2005).
Species
Reproductive mode
Habitat for reproduction
Conservation status
Family: Centrolenidae (7 spp.)
Mode 25: Eggs hatching into
exotrophic tadpoles that drop in
lotic water
Centrolene ballux
Mode 25
Vegetation along fast-flowing
streams
Critically Endangered
Centrolene heloderma
Mode 25
Vegetation along fast-flowing
streams
Critically Endangered
Centrolene lynchi
Mode 25
Vegetation along fast-flowing
streams
Endangered
Centrolene peristictum
Mode 25
Vegetation along fast-flowing
streams
Vulnerable
Nymphargus griffithsi
Mode 25
Vegetation along fast-flowing
streams
Vulnerable
Nymphargus grandisonae
Mode 25
Vegetation along fast-flowing
streams
Least Concern
Nymphargus lasgralarias
Mode 25
Vegetation along fast-flowing
streams
Data Deficient
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Batrachochytrium dendrobatidis in an Andean frog community
Appendix 2 (continued). Amphibians at Reserva Las Gralarias, with corresponding lUCN (2014) conservation status. The list
includes three potential new species (Pristimantis sp. 1, Pristimantis sp. 2, and Pristimantis sp. 3). Reproductive modes are sensu
Haddad and Prado (2005).
Species
Reproductive mode
Habitat for reproduction
Conservation status
Family: Craugastoridae (16
spp.)
Mode 23: Direct development of
terrestrial eggs
Pristimantis achatinus
Mode 23
Terrestrial, mainly in pastures and
modified environments
Least Concern
Pristimantis appendiculatus
Mode 23
Terrestrial, mainly primary and
secondary forests
Least Concern
Pristimantis calcarulatus
Mode 23
Terrestrial, mainly primary and
secondary forests
Vulnerable
Pristimantis crenunguis
Mode 23
Terrestrial, mainly primary and
secondary forests
Endangered
Pristimantis eremitus
Mode 23
Terrestrial, mainly primary and
secondary forests
Vulnerable
Pristimantis eugeniae
Mode 23
Terrestrial, mainly primary and
secondary forests
Endangered
Pristimantis hectus
Mode 23
Terrestrial, mainly primary and
secondary forests
Data deficient
Pristimantis illotus
Mode 23
Terrestrial, mainly primary and
secondary forests
Near Threatened
Pristimantis parvillus
Mode 23
Terrestrial, mainly primary and
secondary forests
Least Concern
Pristimantis pteridophilus
Mode 23
Terrestrial, mainly primary and
secondary forests
Endangered
Pristimantis sobetes
Mode 23
Terrestrial, mainly primary and
secondary forests
Endangered
Pristimantis verecundus
Mode 23
Terrestrial, mainly primary and
secondary forests
Vulnerable
Pristimantis w-nigrum
Mode 23
Terrestrial, mainly primary and
secondary forests
Least Concern
Pristimantis sp. 1
Mode 23
Terrestrial, mainly primary and
secondary forests
Not evaluated
Pristimantis sp. 2
Mode 23
Terrestrial, mainly primary and
secondary forests
Not evaluated
Pristimantis sp. 3
Mode 23
Terrestrial, mainly primary and
secondary forests
Not evaluated
Family: Hylidae (3 spp)
Dendropsophus carnifex
Mode 1 : Eggs and exotrophic
tadpoles in lentic water
Ponds
Least Concern
Hyloscirtus alytolylax
Mode 25
Vegetation along fast-flowing
streams
Near Threatened
Hyloscirtus criptico
Mode 25
Vegetation along fast-flowing
streams
Not evaluated
Family: Caeciliidae (1 sp.)
Caecilia buckleyi
Family: Rhinatrematidae (1 sp.)
Unknown
Unknown
Not evaluated
Epicrionops bicolor
Unknown
Unknown
Least Concern
August 2014 | Volume 8 | Number 1 | e81
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Guayasamin et al.
Juan M. Guayasamin is head of the Centro de Investigacion de la Biodiversidad y Cambio Climatico (Bio-
Camb) and Professor at Universidad Tecnologica Indoamerica, Quito, Ecuador. He obtained his Master's de-
gree and Ph.D. in Ecology and Evolutionary Biology (University of Kansas, EEUU). His research includes am-
phibian phylogenetic systematics, taxonomy, biogeography, ecology, and conservation biology {Photographed
by Lucas Bustamante).
Angela M. Mendoza is a biologist from Universidad del Valle (Colombia) with a Master’s in biological sci-
ences at the Universidad Nacional Autonoma de Mexico (UNAM). She is a research assistant in the Conserva-
tion Genetics Laboratory at the Alexander von Humboldt Institute, Colombia. Her main interest is to apply
molecular tools in ecology and conservation, with emphasis in terrestrial vertebrates, mainly Neotropical am-
phibians {Photographed by Angela M. Mendoza).
Ana V. Longo is a doctoral student in the Department of Ecology and Evolutionary Biology at Cornell Uni-
versity, Ithaca, New York. Her main interests are amphibian disease ecology and evolution. Her research aims
to understand the mechanisms underlying seasonal and ontogenetic patterns of disease susceptibility in direct-
developing frogs {Photographed by Alberto L. Lopez-Torres).
Kelly R. Zamudio is a Professor in the Department of Ecology and Evolutionary Biology at Cornell Univer-
sity. She received her B.A. from UC Berkeley in Zoology in 1991, and her Ph.D. from University of Washing-
ton, Seattle, in 1996. Her research focuses on the origin and maintenance of vertebrate biodiversity (especially
reptiles and amphibians). Her lab integrates field research in population biology, demography, and habitat
change with lab research on the genomic underpinnings of population diversification, speciation, and conserva-
tion. {Photographed by Karen Lips).
Elisa Bonaccorso is a biologist, Ph.D. in Ecology and Evolutionary Biology (University of Kansas, EEUU),
and Licenciada en Biologia (Universidad Simon Bolivar, Venezuela). Her research is broad and includes mo-
lecular systematics, ecology of wildlife diseases, and conservation biology {Photographed by Juan M. Guayas-
amin).
Amphib. Reptile Conserv. | amphibian-reptile-conservation.org (44)
August 2014 | Volume 8 | Number 1 | e81
Anolis podocarpus. Photo by A. Almenddriz.
September 2014 | Volume 8 | Number 1
Amphib. Reptile Conserv. | amphibian-reptile-conservation.org (45)
e82
Amphibian & Reptiie Conservation
8(1) [Special Section]: 45-64.
Copyright: © 2014 Almendariz et al. This is an open-access article distributed under the
terms of the Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported
License, which permits unrestricted use for non-commercial and education purposes only
provided the original author and source are credited. The official publication credit source:
Amphibian & Reptile Conservation at: amphibian-reptile-conservation.org
Overview of the herpetofauna of the unexplored
Cordillera del Condor of Ecuador
^Ana Almendariz, ^John E. Simmons, ^ ^*Jorge Brito, and ^’^Jorge Vaca-Guerrero
^Instituto de Ciencias Biologicas, Escuela Politecnica Nacional, Casilla 17-01-2759, Quito, ECUADOR ^Museologica, 128 Burnside Street,
Bellefonte, Pennsylvania 16823 USA
Abstract. — ^The Cordillera del Condor is an area rich in unique vegetation assemblages and endemic
faunal elements; the herpetofauna is especially diverse, particularly the anurans. The montane
forest and sandstone tepuis, located atop large andesite and quartz formations, provide a variety
of habitats and microhabitats in which the herpetofauna finds food, shelter, and reproductive sites,
such as terrestrial and arboreal bromeliads and a soil type termed “bamba” that is covered with
mosses and roots. Information compiled from publications and recent studies has revealed the
presence of 120 species of amphibians and 59 species of reptiles, including 41 probable new species
(36 amphibians and five reptiles) in the genera Centrolene, Dendrobates, Pristimantis, Lynchius,
Chiasmocieis, Boiitogiossa, Anoiis, Erythroiamprus, Tantilia, and Dipsas.
Resumen. — La Cordillera del Condor es un area rica en formaciones vegetales unicas y elementos
faum'sticos endemicos; presenta una singular diversidad herpetofaunistica, particularmente de la
anurofauna. Los bosques montanos y los de “tepuy,” asentados sobre piedras grandes de andesita
y cuarzo crean variedad de habitats y microhabitats, en donde la herpetofauna encuentra alimento,
refugio y lugares para la reproduccion, como por ejempio las bromelias terrestres y arboreas y
un suelo denominado “bamba” que esta cubierto de musgos y raices. La informacion recopilada
de material publicado y de los estudios realizados en los ultimos ahos revela la presencia de 120
especies de anfibios y 59 especies de reptiles. Los resultados incluyen 41 especies posiblemente
nuevas (36 anfibios y cinco reptiles) de los generos: Centroiene, Dendrobates, Pristimantis,
Lynchius, Chismocieis, Boiitogiossa, Anoiis, Erythroiamprus, Tantiiia, y Dipsas.
Key words. Ecuador, Cordillera del Condor, amphibian, reptile
Citation: Almendariz A, Simmons JE, Brito J, Vaca-Guerrero J. 2014. Overview of the herpetofauna of the unexplored Cordillera del Condor of Ecuador.
Amphibian & Reptile Conservation 8(1 ) [Special Section]: 45-64 (e82).
Introduction
The fauna of Ecuador, in general, has not been extensive-
ly studied, despite a notable increase in research activ-
ity in recent years (Albuja et al. 2012). In particular, the
Cordillera del Condor region, in southern Ecuador along
the border with Pern (Figure 1), is a very poorly known
area. The purpose of this paper is to suncnnarize and re-
view herpetofaunal studies of the Cordillera del Condor
region. Studies of the avian and mammalian fauna have
been published elsewhere (e.g., Albuja and Patterson
1996; Brito and Arguero 2012; Freile et al. 2014).
The long-running border conflicts between Ecuador
and Pern and the difficulty in accessing the region have
maintained the ecosystems of the Cordillera del Condor
almost intact. It has only been since the end of the con-
flicts known as the Pasquisha War (which ended in Feb-
mary 1981) and the Alto Cenepa War (which ended in
Febmary 1995), that roads into the area have begun to
open, which has resulted in incipient colonization and an
awakened interest in mineral prospection in the region.
Nevertheless, there are still some parts of the Cordillera
del Condor that remain unaltered.
The Cordillera del Condor is part of a biologically di-
verse, discontinuous, sub-Andean cordillera that has sev-
eral characteristics that distinguish it from the rest of the
Andes. Whereas the main Andes are of metamorphic and
igneous origin, the Cordillera del Condor is sedimentary,
composed largely of limestone and sandstone (Schul-
enberg and Awbrey 1997). The region is dominated by
*Current address: Museo Ecuatoriano de Ciencias Maturates, Rumi-
pamba y Avenida de los Shyris, Quito, ECUADOR
Correspondence. Emails: ^ ana. almendariz® epn.edu.ee, ^simmons.johne® gmail.com (Corresponding author, John E. Sim-
mons), ^jorgeyakuma® yahoo.es, "^gheovak® hotmail.com
Amphib. Reptile Conserv. | amphibian-reptile-conservation.org (46) September 2014 | Volume 8 | Number 1 | e82
Almendariz et al.
i
77-D'O-W
■—
CD
_|5
^Hivnpabhokn
COLOMBIA
ECUADOR
1 1
ECUAI>0|SE
6 fKta r Mr 1 d Of
1 1 PEHU
yiadiriizA
tA
■k
^in rungirtzi PERU
0 25
50
100
-
-
TS-o'™
T7-o'o'W
Fig. 1. Map of the Cordillera del Condor region.
geologically complex mountains topped with sandstone
plateaus at elevations of 300 m to almost 3,000 m that
support habitats similar to the sandstone mountains of
the Guyana Shield (Figure 2); many of the plateaus have
vegetation similar to that of the tepuis. Due to its loca-
tion just northeast of the Huancabamba Depression, the
lowest point in the Andes (Duellman 1999), the Cordil-
lera del Condor receives moisture from both the Atlantic
and the Pacific sides of the Andes — moisture from the
Western slopes of the Andes as well as moisture moving
east across the Amazon basin drops over the Cordillera
del Condor, providing frequent, year-round precipitation
(Schulenberg and Awbrey 1997).
The first systematic botanical studies of the region
were carried out in 1990 and 1991 in the Rfo Nangaritza
basin (in the southern region of the Cordillera del Con-
dor), under the auspices of the Proyecto Promobot and
the Tratado de Cooperacion Amazonica, with the partici-
pation of both Ecuadorian and foreign scientists. These
explorations were limited to areas below about 1,700 m
Fig. 2. Alto Paquisha, 2,400 m. Photo by A. Almendariz.
in altitude. In 1993, A. H. Gentry collected plants on one
of the highest points of the mountain range (2,100 m)
as part of a Rapid Assessment Program (RAP) survey,
organized by Conservation International (Cl, a non-gov-
ernmental organization). Gentry found that the vegeta-
tion structure and families of plants were similar to those
of the sandstone tepuis of the Guiana Highlands (Schul-
enberg and Awbrey 1997). In recent years, D. Neill and
his collaborators have surveyed the flora at several points
in the cordillera, publishing descriptions of new species
and studying the environmental heterogeneity associated
with variable types of soils (Neill 2005; Neill and Asan-
za 2012; Neill and Ulloa 2011; Riina et al. 2014; Ulloa
et al. 2012). The diversity of plant assemblages on the
sandstone plateaus produces a variety of microhabitats
that provide food, shelter, and reproductive sites for the
herpetofauna, particularly terrestrial and arboreal brome-
liads; the “bamba” soils of many of these tepuis is thickly
covered with mosses and roots, and serves to Alter the
tannins that darken the turbid water in creeks and streams
(Figure 3).
According to the ecosystem classification for conti-
nental Ecuador (Ministerio del Ambiente del Ecuador
2012), the following ecosystems have been identified in
the Cordillera del Condor:
• Evergreen piedmont forest in the Condor-Kutuku
ranges
• Evergreen forest on the sandstone plateaus of the
Condor range in the lower Ecuadorian Amazon
Fig. 3. Vegetation in the interior of a tepui forest. Photo by A.
Almendariz.
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September 2014 | Volume 8 | Number 1 | e82
Herpetofauna of the Cordillera del Condor of Ecuador
• Lower montane evergreen forest in the Condor-
Kutuku ranges
• Evergreen piedmont forest on the sandstone mesas
of the Condor-Kutuku ranges
• Evergreen lower montane forests on the sandstone
mesas of the Condor-Kutuku ranges
• Montane humid shrub in the Condor range
• Evergreen montane forests on the sandstone mesas
of the Condor-Kutuku ranges
• Montane humid shrub with herbaceous rosette
thickets (herbazales) in the Condor range
Materials and Methods
Herpetological surveys of the region have been few and
limited (Figure 1). The information presented below is
drawn from an extensive survey of the literature and
recent field work. The majority of the studies have em-
ployed the Rapid Ecological Assessment strategy or RAP
developed by Cl (Sayre et al. 2002), in habitats where the
presence of herpetofaunal elements was anticipated.
The northern zone of the Cordillera del Condor is
known from three studies. The first was carried out in
1972 in conjunction with a privately funded orchid col-
lecting expedition (accompanied by personnel from the
Missouri Botanical Garden and the University of Kansas
Museum of Natural History), at elevations of 870-2,000
m at the headwaters of the Rio Piuntza, Rio Chuchum-
bleza, Rio Numpatacaimi, and Rio Santa Agueda in Mo-
rona Santiago Province (Duellman and Simmons 1988).
The second survey was a RAP assessment conducted by
Cl, the Escuela Politecnica Nacional, Fundacion Fedima,
and the Universidad Nacional Mayor de San Marcos.
The areas surveyed included the Ecuadorian flank of the
Cordillera del Condor (Coangos and Achupallas in Mo-
rona Santiago Province [Figure 4], Miazi and Shaimi in
Zamora Chinchipe Province). The Peruvian flank of the
Cordillera del Condor was surveyed at the base of Cerro
Machinaza, Alfonso Ugarte-PV3, Falso Paquisha-PV22,
and Puesto de Vigilancia Comainas. Subsequently, a
third survey was carried out by Fundacion Natura (FN
2000) to establish the Parque El Condor, which inven-
Fig. 4. Achupallas sector, 2,100 m. Photo by A. Almenddriz.
toried the Comunidad Numpatakaime and confluence of
the Rio Tsuirim and the Rio Coangos.
Another survey, conducted as part of the Proyecto Paz
y Conservacion Binacional en la Cordillera del Condor
Ecuador-Peru by the Organizacion Intemacional de las
Maderas Tropicales, Conservation International, Fun-
dacion Natura, and the Institute Nacional de Recursos
Naturales (INRENA) in 2005 (Organizacion Intemacio-
nal de las Maderas Tropicales and Fundacion Natura y
Conservacion Intemacional 2005), collected data from
several localities in the southern sector on the Ecuadorian
flank of the Cordillera del Condor, including Condor Mi-
rador and Herradura. The corresponding Pemvian flank
survey was focused on the Zona Reservada Santiago Co-
maina.
Between March 2008 and July 2012, the Escuela Poli-
tecnica Nacional team, under a contract with the Cardno-
Entrix Corporation, carried out 16 expeditions to survey
the herpetofauna of Alto Manchinaza. In 2009, a Cl RAP
survey was conducted by personnel from the Pontificia
Universidad Catolica del Ecuador, Louisiana State Uni-
versity, and Fundacion Ecologica Arcoiris, with support
from Secretaria Nacional de Ciencia y Tecnologia del
Ecuador (SENACYT) of the tepuyes of the upper basin
of the Rio Nangaritza (Guayasamm et al. 2011). In 2012,
the Fundacion Naturaleza y Cultura Intemacional and the
Universidad Estatal Amazonica organized an expedition
to Cerro Plateado (Figure 5), the southern point of the
Cordillera del Condor, which included researchers from
the Escuela Politecnica Nacional.
Results
The 1972 survey of the northern zone of the Cordillera
del Condor resulted in the capture of 30 species, includ-
ing nine new species (e.g., Duellman and Simmons 1988,
Lynch 1974, 1976, 1979; Lynch and Duellman 1980).
Specimens and additional records from this survey are
deposited in the Biodiversity Research Institute at the
University of Kansas, along with additional related spec-
imens accounting for 47 species total (Reynolds 1997;
Schulenberg and Awbrey 1997).
Fig. 5. Peak of Cerrro Plateado, 2,900 m. Photo by V. Carvajal.
Amphib. Reptile Conserv. | amphibian-reptile-conservation.org (48)
September 2014 | Volume 8 | Number 1 | e82
Almendariz et al.
Fig. 6. Pristimantis sp. Photo by A. Almendariz.
Fig. 7. Centrolene condor. Photo by A. Almendariz.
Fig. 8. Excidobates condor. Photo by A. Almendariz.
Fig. 9. Enyalioides rubrigularis (female). Photo by A. Fig. 10. Hyloscirtus condor. Photo by J. Brito.
Almendariz.
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September 2014 | Volume 8 | Number 1 | e82
Herpetofauna of the Cordillera del Condor of Ecuador
The second survey of the northern Zone of the Cordil-
lera del Condor recorded a total of 34 species — 27 am-
phibians and seven reptiles (Almendariz 1997a, 1997b).
Although the survey was conducted under adverse en-
vironmental conditions, geographic range extensions for
several species were recorded. The Peruvian flank sur-
veys recorded 58 species (35 anurans and 23 reptiles);
the data from the Peruvian surveys was collected by per-
sonnel from the Museo de Historia Natural of the Univer-
sidad Nacional Mayor de San Marcos in 1987 (Reynolds
and Icochea 1997a, 1997b) and expanded the ranges of
two species for Peru, Rhinella festae and Hemiphractus
bubalus. The Parque El Condor survey lists a total of 36
species (22 anurans, eight lizards, and six snakes), in-
cluding nine species new to the Cordillera del Condor
herpetofauna, and makes reference to the difficulty of
identifying some of the material. The report summarizes
information known up to the year 2000, and lists a total
of 95 species for the region (56 amphibians and 39 rep-
tiles).
The survey of the southern sector of the Cordillera del
C6ndor collected specimens that were not identified to
the species level of the genus Pristimantis (Figure 6), and
a glass frog provisionally identified as Centrolene cf. cro-
ceopodes that in 2008 was named as Centrolene condor
(Cisneros-Heredia and Morales-Mite 2008; Figure 7).
The surveys of Alto Manchinaza recorded 70 species
of amphibians and 43 species of reptiles (Almendariz et
al. in prep.). The results of these surveys revealed the
Fig. 11. Herpetofaunal assemblages and endemics from the
Cordillera del Condor.
ua UO
AMPHSBIANS RtPHlLES
□ TdIhI N*5ppcl^ M Pntpntialty Nfw Speaps
Fig. 12. Documented and predicted species diversity in the
Cordillera del Condor.
Amphib. Reptile Conserv. | amphibian-reptile-conservation.org (50)
presence of several little known or new species, includ-
ing a new species of highland poison dart frog, Excido-
bates condor (Almendariz et al. 2012; Figure 8) and geo-
graphic range extensions for Phyllomedusa ecuatoriana,
Centrolene condor, Chimerella mariaelenae, Hyloxalus
mystax, and Enyalioides rubrigularis (Figure 9), among
others. In addition, ecological data and information on
reproduction, vocalizations, and other aspects of the life
history for these species was collected (Almendariz and
Batallas 2012a, 2012b; Batallas and Brito 2014; Brito et
al. in prep.); at least 28 probably new species from differ-
ent genera {Centrolene, Bolitoglossa, Pristimantis, Ano-
lis, Atractus, Erythrolamprus, Tantilla, and Dipsas) were
obtained, which are in the process of being described.
This project included environmental education and com-
munity outreach work (Almendariz 2012).
The 2009 Pontificia Universidad Catolica del Ecua-
dor survey recorded 27 species of amphibians and 17
reptiles, including a new species of anuran, Pristiman-
tis minimus (Teran- Valdez and Guayasamm 2010). This
project included the publication of a field guide to plants
and animals of the tepuis of Nangaritza (Almendariz
2010; Freile et al. 2010).
The 2012 expedition to Cerro Plateado (Almendariz
and Brito 2013) recorded 19 species. Of these, 14 were
anurans and salamanders, including nine species of the
family Craugastoridae (most were members of the genus
Pristimantis). Based on the ecology of the area, it is as-
sumed that more species occur at this locality, including
members of the family Centrolenidae. A new species of
torrent frog, Hyloscirtus condor (Figure 10), was de-
scribed based on specimens obtained on this expedition
(Almendariz et al. 2014), and at least eight new species
in the genera Lynchius, Pristimantis, and Bolitoglossa
were obtained that will be described in the future.
Discussion
The Cordillera del Condor is of particular importance
due to its high biodiversity and the presence of several
unique ecosystems (e.g., the sandstone formations simi-
lar to tepuis). The Condor region, with its diverse range
of habitats, contains numerous species that correspond to
three faunal components: (1) Amazonian lowlands; (2)
eastern flanks of the Andes, and (3) an endemic fauna
limited to the southern part of Ecuador (Duellman and
Lynch 1988). As shown in Figure 11, the Baja Amazonia
herpetofaunal assemblage contains more reptile species
(63%) than amphibian species (28%). In the herpetofau-
nal assemblages associated with the eastern slopes of the
Andes there are slightly more amphibian species (34%
and 32%). The percentages relative to the endemic com-
ponent of the Cordillera del Condor are notably higher in
amphibians than in reptiles (41% and 7%).
A summary of the herpetofaunal diversity of the
region is provided in Tables 1 and 2; a comparison of
known species diversity and predicted species diversity
September 2014 | Volume 8 | Number 1 | e82
Almendariz et al.
Fig. 13. Lynchius sp. Photo by J. Brito.
Fig. 15. Cercosaura dicra. Photo by G. Gallardo.
Fig. 14. Pristimantis muscosus. Photo by A. Almendariz.
Fig. 16. Erythrolamprus sp. Photo by A. Almendariz.
Fig. 17. Tantilla sp. Photo by A. Almendariz.
Fig. 18. Anolis podocarpus. Photo by A. Almendariz.
for the region is provided in Figure 12. Some of the more
distinctive species found in the region include frogs of
the genera Lynchius (Craugastoridae; Figure 13) and
Pristimantis (Craugastoridae; Figure 14), the gynmoph-
thalmid lizard Cercosaura dicra (Figure 15), and the
colubrid snakes Erythrolamprus (Figure 16) and Tantilla
(Figure 17).
During the last five years, the following species have
been described based on material from the Cordillera del
Condor: Enyaliodes rubrigularis (Torres-Carvajal et al.
2009; Figure 9), Anolis podocarpus (Ay ala- Varela and
Torres-Carvajal 2010; Figure 18), Pristimantis minimus
(Teran- Valdez and Guayasanun 2010), Excidobates con-
dor (Almendariz et al. 2012; Figure 8), Hyloscirtus con-
dor (Almendariz et al. 2014; Figure 10), and Siphlophis
ayauma (Sheehy et al. 2014). The work has expanded the
known geographic distribution of Anolis soini (Ayala-
Varela et al. 2011; Figure 19) and revealed new distribu-
Amphib. Reptile Conserv. | amphibian-reptile-conservation.org (51)
September 2014 | Volume 8 | Number 1 | e82
Herpetofauna of the Cordillera del Condor of Ecuador
tion and natural history information for two other species
of the genera Centrolene and Hyloxalus (Almendariz and
Batallas 2012a, 2012b). It is also noteworthy that several
new species are in the process of being described in the
genera Pristimantis and Chiasmocleis (Almendariz et al.
in prep.). A detailed publication about the herpetofauna
of the Condor region is in preparation (Almendariz et al.
in prep.).
Based on information published in the most recent
studies conducted in the Cordillera del Condor, there are
a total of 120 amphibian species (11 families, 31 gen-
era), and 59 reptile species (nine families, 28 genera), not
including those found at elevations below 850 m (Fig-
ure 20). In addition, based on the specimens discussed
above, the area contains approximately 41 potentially
new species (36 amphibians and five reptiles; see Figure
12). These numbers indicate that the region has signifi-
cant endemic diversity (see Table 1, Table 2, and Figure
11 ).
Conclusion
Within the Cordillera del Condor, four areas protected by
the Sistema Nacional de Areas Protegidas have been es-
tablished: (1) Reserva Biologica El Condor; (2) Reserva
Biologica El Quimi; (3) Reserva Biologica Cerro Platea-
do; and (4) Refugio de Vida Silvestre El Zarza. On the
eastern flank, the Peruvian government has concentrated
its efforts to create Parque Nacional Ichigkat Muja-Cor-
dillera del Condor (SERNANP 2012). Nevertheless, the
ecosystems in the Cordillera del Condor are threatened
by imminent human colonization and settlement, the in-
troduction of agriculture and livestock, and mining; the
latter activity poses the greatest threat to the conserva-
tion of the tepui-like forests and the health of the aquatic
ecosystems, which are the reproductive habitats of many
species of anurans, including hylids and centrolenids. On
the other hand, the fact the amphibians of the Andean re-
gion have limited distribution makes them susceptible to
extinction, and in some cases, the protection of their hab-
itat does not improve their chance of survival (Guayas-
amin et al. 2011). This situation warrants intensified
research and conservation studies of these vertebrates,
especially in little explored areas as in the case of the
Cordillera del Condor. The preliminary results of surveys
of Alto Machinaza and Cerro Plateado have revealed the
presence of possibly new species in these areas, indicat-
ing that future interventions in these areas should comply
strictly with the measures to protect ecosystems, environ-
mental mitigation, and management plans.
Acknowledgments. — We thank the Kinross and Card-
no-Entrix Corporation and their administrative and field
staff for use of facilities to carry out recent held stud-
ies, and Eundacion Naturaleza y Cultura Internacional
and David Neill of the Universidad Estatal Amazonica
for the invitation to participate in the expedition to Cerro
Fig. 19. Anolis soini. Photo by J. Vaca G.
Fig. 20. Familial, generic, and specific diversity of amphibians
and reptiles in the Cordillera del Condor.
Plateado. Thanks also to Bruce MacBryde and the late
Milan D. Eiske for the opportunity to participate in the
1972 expedition into the Cordillera del Condor.
Literature Cited
Albuja L, Almendariz A, Barriga R, Montalvo ED, Ca-
ceres E, Roman JS. 2012. Fauna de Vertebrados del
Ecuador. Instituto de Ciencias Biologicas, Escuela
Politecnica Nacional, Quito Ecuador.
Albuja L, Patterson B. 1996. A new species of northern
shrew-opossum (Paucituberculata: Caenolestidae)
from the Cordillera del Condor, Ecuador. Journal of
Mammalogy 77: 41-53.
Almendariz A. 1997a. Reptiles and amphibians of the
Cordillera del Condor. Pp. 80-82 In: The Cordillera
del Condor Region of Ecuador and Peru: A Biologi-
cal Assessment. Editors, Schulenberg T, Awbrey K.
RAP Working Papers number 7, Conservation Inter-
national, Washington, D.C., USA. 234 p. Available:
https://library.conservation.org/Published%20Docu-
ments/2009/07%20RAP%20Working%20Papers.pdf
[Accessed: 30 August 2014].
Almendariz A. 1997b. Amphibian and reptile species
recorded in the Northern and Western Cordillera del
Condor. Pp. 199-201 In: The Cordillera del Con-
dor Region of Ecuador and Peru: A Biological As-
Amphib. Reptile Conserv. | amphibian-reptile-conservation.org (52)
September 2014 | Volume 8 | Number 1 | e82
Table 1. Species of amphibians recorded from the Cordillera del Condor.
Almendariz et al
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Amphib. Reptile Conserv. | amphibian-reptile-conservation.org (54)
September 2014 | Volume 8 | Number 1 | e82
Almendariz et al
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Amphib. Reptile Conserv. | amphibian-reptile-conservation.org (55)
September 2014 | Volume 8 | Number 1 | e82
Herpetofauna of the Cordillera del Condor of Ecuador
Rio
Puintza
Duellman
& Lynch
1988
1550-1830
msnm
X
RAP 7
Reyn-
olds &
Icochea
1997
665-1750
msnm
RAP 7
Al-
mendariz
1997
900-2200
msnm
X
X
Nump-
atkaim
/ rios
Tsuirim y
Coangos
Fun-
dacion
Natura
2000
930-1050
msnm
X
Quebrada
Shinga-
natza
Proy.
Paz y
Conserva-
cion, 2005
(Peru)
850-1200
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Amphib. Reptile Conserv. | amphibian-reptile-conservation.org (56)
September 2014 | Volume 8 | Number 1 | e82
Almendariz et al
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Amphib. Reptile Conserv. | amphibian-reptile-conservation.org (57)
September 2014 | Volume 8 | Number 1 | e82
Herpetofauna of the Cordillera del Condor of Ecuador
Rio
Puintza
Duellman
& Lynch
1988
1550-1830
msnm
RAP 7
Reyn-
olds &
Icochea
1997
665-1750
msnm
X
X
RAP 7
Al-
mendariz
1997
900-2200
msnm
X
X
X
Nump-
atkaim
/ rios
Tsuirim y
Coangos
Fun-
dacion
Natura
2000
930-1050
msnm
X
Quebrada
Shinga-
natza
Proy.
Paz y
Conserva-
cion, 2005
(Peru)
850-1200
msnm
c/3
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Amphib. Reptile Conserv. | amphibian-reptile-conservation.org (58)
September 2014 | Volume 8 | Number 1 | e82
Table 2 . Species of amphibians recorded from the Cordillera del Condor.
Almendariz et al
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Amphib. Reptile Conserv. | amphibian-reptile-conservation.org (59)
September 2014 | Volume 8 | Number 1 | e82
Herpetofauna of the Cordillera del Condor of Ecuador
oQ
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Amphib. Reptile Conserv. | amphibian-reptile-conservation.org (61)
September 2014 | Volume 8 | Number 1 | e82
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Received: 19 May 2014
Accepted: 20 August 2014
Published: 20 September 2014
Amphib. Reptile Conserv. | amphibian-reptile-conservation.org (63)
September 2014 | Volume 8 | Number 1 | e82
Herpetofauna of the Cordillera del Condor of Ecuador
Ana Almendariz is a researcher and the Curator of Herpetology at the Institute of Biological Sciences at the
Escuela Politecnica Nacional in Quito, Ecuador. A native of Quito, Almendariz holds an undergraduate degree
in biology and a master’s degree in conservation and management of natural resources. She conducts research
on amphibians and reptiles throughout Ecuador and has published extensively on her research.
John E. Simmons is president of Museologica consulting, and teaches museum studies for Kent State Uni-
versity, Juniata College, and the Universidad Nacional de Colombia. Simmons has an undergraduate degree in
systematics and ecology and a master’s degree in historical administration and museum studies, and previously
was collections manager at the California Academy of Sciences and the Biodiversity Research Institute at the
University of Kansas.
Jorge Brito is a mammal and amphibian researcher at the Museo Ecuatoriano de Ciencias Naturales in Quito,
Ecuador. He has an undergraduate degree in biology from the Universidad Central del Ecuador; his research
interests are focused on amphibians and terrestrial micromammals. He has published several contributions on
these species principally from southeastern Ecuador.
Jorge Vaca-Guerrero is a Junior Investigator at the Institute de Ciencias Biologicas of the Escuela Politec-
nica Nacional in Quito, Ecuador. He has an undergraduate degree in biology from the Universidad Central del
Ecuador, and experience in studies of the herpetofauna of the Eastern region of Ecuador. His principle area of
interest is the evolution of reptiles, particularly vipers.
Amphib. Reptile Conserv. | amphibian-reptile-conservation.org (64)
September 2014 | Volume 8 | Number 1 | e82
Copyright: © 2014 McCracken and Forstner. This is an open-access article distributed
under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs 3.0
Unported License, which permits unrestricted use for non-commercial and education pur-
poses only provided the original author and source are credited. The official publication
credit source: Amphibian Reptile Conservation at: amphibian-reptile-conservation.org
Amphibian & Reptiie Conservation
8(1) [Special Section]: 65-75.
Herpetofaunal community of a high canopy tank bromeliad
{Aechmea zebrina) in the Yasuni Biosphere Reserve of
Amazonian Ecuador, with comments on the use of
“arboreai” in the herpetologicai iiterature
^’^Shawn F. McCracken and ^’^Michael R. J. Forstner
^Department of Biology, Texas State University, San Marcos, Texas, USA
Abstract . — ^Tank bromeliads provide microhabitat that supports a high diversity of organisms in
the harsh environment of tropical forest canopies. Most studies of organisms occupying tank
bromeliads have focused on invertebrates found within bromeliads near or at ground level. Few
investigations of vertebrate communities utilizing this keystone resource are available. We describe
the amphibian and reptile community occupying the high canopy tank bromeliad, Aechmea zebrina,
in lowland rainforest of the Yasum' Biosphere Reserve in the Amazon Basin of Ecuador. We used
single-rope climbing techniques to sample a total of 160 A. zebrina bromeliads from 32 trees, at
heights of 18.3 to 45.5 m above ground. We collected 10 metamorphosed anuran species, one gecko,
one snake, and observed two species of lizard within bromeliads. Summary statistics for a suite
of environmental factors associated with herpetofauna in A. zebrina bromeliads are reported. We
estimated the density of anurans occupying A. zebrina communities and contrast these estimates
with anuran densities from tropical forest floor anuran studies. Finally, we discuss the use of the
term “arboreal” within the herpetologicai literature, and make recommendations for terminology
used to describe the vertical space occupied by a species or assemblage.
Key words. Amphibian, anuran, epiphyte, forest, microhabitat, rainforest, reptile
Citation: McCracken SF, Forstner MRJ. 2014. Herpetofaunal community of a high canopy tank bromeliad {Aechmea zebrina) in the Yasuni Biosphere
Reserve of Amazonian Ecuador, with comments on the use of “arboreal” in the herpetologicai literature. Amphibian & Reptile Conservation 8(1) [Special
Section]: 65-75 (e83).
Introduction
Forest canopies provide habitat for approximately 50%
of terrestrial species, yet there are few studies specific
to canopy herpetofauna (Stewart 1985; Vitt and Zani
1996; Kays and Allison 2001; Guayasamin et al. 2006;
McCracken and Forstner 2008; Lowman and Schowal-
ter 2012; Scheffers et al. 2013; McCracken and Forstner
2014). Basic ecological knowledge of arboreality (tree-
living) and utilization of high canopy microhabitats by
amphibians and reptiles remain depauperate in the litera-
ture (Moffett 2000; Kays and Allison 2001; Lehr et al.
2007). A canopy microhabitat frequently used by herpe-
tofauna in tropical forests are epiphytes, and in particular
epiphytic tank bromeliads that are phytotelms capable of
holding relatively large volumes of water (Lowman and
Rinker 2004; McCracken and Forstner 2008). In lowland
Neotropical rainforest, canopy tank bromeliads typically
reside in the overstory and emergent canopy trees at ver-
tical heights of 5^5 -f meters with ~5 to >150 individuals
on a single tree (McCracken and Forstner 2006). These
arboreal bromeliad conununities create a three-dimen-
sional “wetland in the sky” that have been estimated to
impound up to 50,000 liters of water per hectare (Kitch-
ing 2000; McCracken and Forstner 2006). Tank bromeli-
ads function as a “keystone resource” in the harsh forest
canopy environment where the atmosphere meets and in-
teracts with 90% of Earth’s terrestrial biomass; providing
a climate-buffered refuge, water source, and food source
for canopy herpetofauna (Nadkami 1994; Ozanne et al.
2003; Cardeliis and Chazdon 2005).
Kays and Allison (2001) found only 4% of 752 arti-
cles published between 1988 and 1998 on tropical forest
arboreal vertebrates focused on reptiles and amphibians.
Many species of herpetofauna are described as being
arboreal regardless of whether they are restricted to the
vertical stratum a few centimeters to a few meters above
ground, or solely inhabit the high forest canopy at 20 or
more meters vertical height (Chaparro et al. 2007; Mc-
Cracken et al. 2007; Guayasamin and Funk 2009). Forest
structure is associated with vertical partitioning or strati-
fication of the component plant community (e.g., trees,
shrubs, lianas) and accentuates vertical patterns followed
by other organisms (Moffett 2000; Lowman and Rinker
Correspondence. Emails: ^smccracken® txstate.edu (Corresponding author); ^ mf@txstate.edu
Amphib. Reptile Conserv. | amphibian-reptile-conservation.org (65)
October 2014 | Volume 8 | Number 1 | e83
McCracken and Forstner
clouds
Roads
Trails
Tree sampled
Yasuni National
Park
20
■ Kilometers
Fig. 1. (A) Map of South America with Ecuador (shaded light blue) and Yasuni National Park (solid dark green) highlighted. The
Amazon ecoregion is outlined with light green line. (B) Northeastern section of Yasuni National Park (light gray line) and surround-
ing region where trees were sampled forAechmea zebrina bromeliads within the vicinity of the Tiputini Biodiversity Station - Uni-
versidad San Francisco de Quito (TBS) and the Yasuni Research Station - Pontificia Universidad Catolica del Ecuador (YRS). (C)
Detail of TBS where trees were sampled for A. zebrina bromeliads. Note: Map is modified from Figure 2 in McCracken and Forstner
(2014) and used under the Creative Commons Attribution license.
2004). Spatial patterns of forest cohabitants, such as tank
bromeliads and their inhabitants, are likewise strongly
influenced by forest structure as a result of the funda-
mental organization of resources and space (Lowman
and Rinker 2004). Identifying the vertical space occupied
by a particular amphibian or reptile species in its given
habitat will allow greater insight to their ecological role
in the system.
Herein, we describe amphibians and reptiles occupy-
ing the high canopy tank bromeliad, Aechmea zebrina, in
lowland rainforest of the Yasum Biosphere Reserve in the
Amazon Basin of Ecuador. We report a suite of environ-
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October 2014 | Volume 8 | Number 1 | e83
Herpetofaunal community of a high canopy tank bromeliad
Fig. 2. (A) A downward vertical view {in situ) of Aechmea zebrina (foreground center left, and at lower elevation in upper right
and center right) and a cluster of Aechmea tessmannii (center, with one in hloom) hromeliads in the tree canopy from ~34 m. (B) A
community of A. zebrina hromeliads at ~38 m (in situ). (C) An A. zebrina hromeliad (ex situ) inside screen tent being measured and
prepared for dismantling, collected from ~44 m in the canopy. Notice the more upright leaves and reddish color because of increased
sun exposure due to high canopy location.
mental factors associated with herpetofauna in A. zebrina
hromeliads. We estimate the density of anurans occupy-
ing mean A. zebrina community sizes in two tree size
classes, representative of our shortest and tallest trees in
the study. We then compared these with anuran densities
from tropical forest floor anuran studies by calculating
the two-dimensional area (m^) of the tree crowns for the
two tree size classes. In completing our review, we feel
it is important to discuss the use of the term “arboreal”
within the herpetological literature and make recommen-
dations for the incorporation of additional terminology
to provide a more informative description of the vertical
space utilized by a species or assemblage.
Materials and Methods
The study was conducted in the northwestern portion of
the Yasum Biosphere Reserve (Yasum) located in Orel-
lana Province, Ecuador. The reserve includes Yasum Na-
tional Park, Waorani Ethnic Reserve, and their respective
buffer and transition zones (Einer et al. 2009). Yasunf is
part of the Napo Moist Eorest terrestrial ecoregion cover-
ing approximately 1 .7 million ha of the upper Amazon
Basin (Einer et al. 2009; Bass et al. 2010). Yasum has an
elevation range of 190-400 m above sea level; the north-
western region averages 2,425-3,145 nun of rainfall per
year with no less than 100 mm per month, temperature
averages 25 °C (15 °-38 °C), and humidity averages 88%
(Blandin 1976; Duellman 1978; Balslev et al. 1987; Bass
et al. 2010). Yasum holds world record species diversity
for several taxa, including the highest documented land-
scape scale (lowland tropical rainforest) herpetofauna di-
versity with 150 species of amphibians and 121 species of
reptiles (Bass et al. 2010). Collections were made in the
vicinity of two research stations, the Tiputini Biodiver-
sity Station (TBS) (0°38’ 14”S, 76°08’60”W) operated by
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October 2014 I Volume 8 I Number 1 I e83
McCracken and Forstner
Fig. 3. A collection of anurans collected from Aechmea zebrina bromeliads. (A) Pristimantis aureolineatus hiding in leaf axil, and
(B) on a leaf of A. zebrina. (C) Pristimantis waoranii emerging from leaf axil, and (D) on a leaf of A. zebrina. (E) Ranitomeya
ventrimaculata and (F) Scinax ruber collected from A. zebrina bromeliads.
the Universidad San Francisco de Quito and the Yasum
Research Station (YRS) (0°40’27”S, 76°23’51”W) oper-
ated by the Pontificia Universidad Catolica del Ecuador
(Fig. 1). Tiputini Biodiversity Station is only accessible
by river and surrounded by undisturbed primary lowland
rainforest, and YRS is located approximately 27 km west
on an oil pipeline road (Maxus road) that has been expe-
riencing forest disturbance within its vicinity but is still
surrounded by large tracts of undisturbed forest. Sam-
pling of A. zebrina bromeliads took place between 0800
and 1800 hours from April to November of 2008.
We focused our sampling on a single large epiphytic
tank bromeliad species, Aechmea zebrina, that is native
to the Amazon regions of Ecuador and southeastern Co-
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October 2014 I Volume 8 I Number 1 I e83
Herpetofaunal community of a high canopy tank bromeliad
lombia (Smith 1953). Aechmea zebrina occupy vertical
heights of approximately 18^5+ m in the overstory and
emergent canopy trees, and range between 1 to >150 in-
dividuals on a single host tree (SFM, unpublished data).
The leaves are upright and arranged in a spiral with their
leaf axils tightly overlapping to form water-holding res-
ervoirs (Fig. 2). These cavities provide a critical refuge
and food source for invertebrate and vertebrate species in
the harsh canopy climate (Nadkami 1994).
Sampling methodology for A. zebrina bromeliads fol-
lowed our previously published methods (McCracken
and Forstner 2008). Single-rope technique (SRT) was
used to climb trees for canopy access, and five brome-
liads were collected haphazardly from each tree at esti-
mated even vertical intervals between one another (Perry
1978). Before each bromeliad removal, we checked for
active amphibians or reptiles, we recorded the bromeli-
ads elevation, measured the air temperature adjacent to
the bromeliad, and the temperature and pH of water held
in one of the outer leaf axils. Ideally, when the bromeliad
is disturbed the response of most animals is a retreat into
the bromeliads leaf bracts and thus prevents loss of speci-
mens (McCracken and Forstner 2008). Bromeliads were
removed and sealed in a 55-gallon (208 L) plastic bag and
then lowered to the ground. After bromeliad collections
we counted the number of A. zebrina inhabiting the tree
and measured tree height. Bromeliads were transported
back to camp where we processed them in a screened tent
to prevent escape of animals. We first poured all water
from the bromeliads through a 1 mm sieve to separate
arthropods, leaf litter, and detritus. We then measured
the water volume with a graduated cylinder and the pH
of the homogenized solution. We counted the number of
mature leaves (used as a size metric) and measured the
height of bromeliads to nearest centimeter (from base of
plant to highest vertical leaf tip). Bromeliads were then
dismantled leaf-by-leaf to collect all herpetofauna.
We identified and counted all metamorphosed an-
urans and reptiles to species level for each bromeliad.
Larval anurans were also collected and counted, with the
majority identified to genus or species. In an attempt to
better identify larval anurans we maintained individual
tadpoles outdoors in 12 oz. plastic cups with water and
detritus collected from bromeliads. Once tadpoles began
to metamorphose the cups were covered with window
screen to prevent escape. Upon sufficient development
to allow identification the froglets were euthanized and
preserved. All herpetofauna were handled and preserved
following the guidelines compiled by the American So-
ciety of Ichthyologists and Herpetologists (ASIH), and
in compliance to the rules overseen by the Texas State
University Animal Care and Use Connnittee (Permit #:
0721-0530-7, 05-05C38ADFDB, and 06-01C694AF).
Additionally, we report the herpetofauna species ob-
served active amongst A. zebrina bromeliads but not
collected. We calculated summary statistics of recorded
habitat variables for each species and report raw data for
Amphib. Reptile Conserv. | amphibian-reptile-conservation.org (69)
Fig. 4. The Banded cat-eyed snake, Leptodeira annulata, col-
lected in an Aechmea zebrina bromeliad at 43.5 m above the
forest floor.
singletons and doubletons. Summary statistics were cal-
culated for recorded habitat variables across all bromeli-
ads sampled, bromeliads occupied by >1 metamorphosed
anurans, and bromeliads not occupied by anurans.
We then compared an estimated number of anuran
individuals in A. zebrina bromeliads per 100 m^ of tree
crown area to other published work of tropical frog as-
semblages collected at or near ground level. No other
studies were available to provide canopy estimates. Mean
anuran abundance per tree was calculated by taking the
mean number of metamorphosed anurans per A. zebrina
bromeliad (x = 0.6) and multiplying by the mean number
of bromeliads per tree (x = 66). Based on tree crown di-
ameter measurements by Asner et al. (2002) in lowland
rainforest of eastern Amazonia we calculated the number
of anurans per 100 m^ of a typical tree crown area for
the two largest tree size classes. The two largest classes
had mean tree heights of 25.3 m (Dominant) and 46.1 m
(Super dominant) with a mean crown diameter of 11.6
m and 19.9 m, respectively. Mean tree crown diameters
were used to calculate the area of a circle. These two tree
size classes were used as we did not measure individual
crown diameters and consider these two as representative
of the shortest (28 m) and tallest (49 m) trees in our study.
We then divided the mean number of anurans per tree
October 2014 I Volume 8 I Number 1 I e83
Water temperature (*C) Bromcliad height (cm) Tree height (m)
McCracken and Forstner
A, zehrina bromdiad host tree height
^ “
O
CTi "
o
1 1 1
Absent Present All Trees
Metamorphosed Anurans
A. zebritia brometiad elevation above
forest floor
1 1 \
Absent Present All Trees
Metamorphosed Anurans
Number of A. zi%riiui bromeliads per tree
o
(N -
A. zebritia bromcLiad heiglit
« e
t
o
o
o
O =
o
w
o
1 1 1
Absent Present All Trees
Metamorphosed Anurans
A, zebrina bromebad leaf number
Absent
Present All Trees
Metamorphosed Anurans
A. z^hritui bromeliad water volume
£
0
1
Absent
Present All Trees
Metamorphosed Anurans
A. zebrina bromeliad vrater temperature
A. zebrina bromeliad water pH
0
1 1 1
Absent Present All Trees
Metamorphosed Anurans
A, zebrina bromeliad water pH (post)
Absent
Present
All Trees
Metamorphosed Anurans
Air temperature at^. zebrina bromeliad
collection site
Fig. 5. Box plots of recorded habitat variables for
Aechmea zebrina bromeliads collected from all trees,
bromeliads with >1 metamorphosed anuran, and bro-
meliads absent of anurans. Asterisks represent the
mean, open circles are outliers, horizontal line inside
box is the median, top and bottom lines of the rectan-
gle are the 3rd and 1st quartiles (Q3 and Ql), respec-
tively, and the top and bottom whiskers are maximum
and minimum values excluding outliers, respectfully.
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October 2014 I Volume 8 I Number 1 I e83
Herpetofaunal community of a high canopy tank bromeliad
in our study by the tree size class crown area calculated
from Asner et al. (2002) and multiplied by 100 to gener-
ate an estimated density of individuals per 100 m^.
All calculations and statistics based on counts of
metamorphosed anurans collected (not larval anurans)
and conducted in the R statistical software (version 3.0.1)
(R Development Core Team 2013).
Results
We sampled five bromeliads from each of 32 trees for a
total of 160 A. zebrina bromeliads sampled. We collected
10 metamorphosed anuran species (Fig. 3), one gecko,
one snake (Fig. 4), and two species of lizard were ob-
served amongst bromeliad leaves but not collected (Ta-
ble 1). A total of 95 metamorphosed anurans (x = 0.6 per
bromeliad) were collected from 56 of the 160 bromeliads
(35%) sampled. Between one and five individuals (x =
1.7), and up to two species were observed in single A. ze-
brina bromeliads occupied by metamorphosed anurans.
The species found together include (number of brome-
liads with species together): Pristimantis aureolineatus
and P. waoranii (7), P waoranii and P acuminatus (1),
P. waoranii and P orphnolaimus (1), P. aureolineatus
and Ranitomeya ventrimaculata (1). We also collected
a minimum of four larval amphibian species from the
water-filled leaf axils of A. zebrina bromeliads includ-
ing Osteocephalus fuscifacies, O. planiceps, Ranitomeya
variabilis, and R. ventrimaculatus. A total of 27 1 larval
anurans were collected from 35 of the 160 bromeliads
(21.9%) sampled, with 14 of the 35 larval occupied bro-
meliads (40%) also occupied by >1 metamorphosed an-
urans. Osteocephalus spp. tadpoles account for 60.5% {n
= 1 64) of confirmed species identifications for all larval
anurans, and these were collected from five bromeliads.
A single O. fuscifacies and a single O. planiceps (both
adults) were each found in separate bromeliads with lar-
vae of same species (identified after rearing). The gecko,
Thecadactylus solimoensis (formerly T. rapicauda), was
found in an A. zebrina bromeliad amongst the outer leaf
axils at 31.5 m above the forest floor in a tree 46.0 m
tall (Bergmann and Russell 2007). The Banded cat-eyed
snake, Leptodeira annulata, was found in a central leaf
axil of an A. zebrina bromeliad at 43.5 m above the for-
est floor in a tree 45.5 m tall (Fig. 4). Anolis transversalis
was observed twice amongst the leaves of A. zebrina bro-
meliads during collections; once on a bromeliad at ~27
m above the forest floor (36 m tall tree) and in another
tree at ~35 m above the forest floor (41 m tall tree). A
single male Anolis ortonii was observed displaying his
dewlap on an outer leaf of an A. zebrina bromeliad at
~20 m above the forest floor in a tree 28 m tall. Summary
statistics for all species reported in Table 1.
Trees sampled for A. zebrina bromeliads were 28 to
49 m in height (x = 40.4 m + 5.5, n = 32), and 28 to 49
m in height (x = 40.2 m + 5.8, n = 27) for trees with >1
bromeliad occupied by metamorphosed anurans. Aech-
mea zebrina bromeliads were collected at above ground
elevations of 18.3 to 44.5 m (x = 32.9 m + 5.6, n = 160),
and bromeliads occupied by >1 metamorphosed anurans
occurred at elevations of 20.5 to 44.5 m (x = 32.1 m +
6.3, n = 56). The number of A. zebrina bromeliads per
host tree was 19 to 150 individuals (x = 66 + 40, n =
32), and 19 to 150 individuals (x = 63 + 38, n = 27) for
trees with >1 bromeliads occupied by metamorphosed
anurans. Aechmea zebrina bromeliads were 45 to 126
cm in height (x = 75 + 14, n = 160), and 51 to 125 cm
in height (x = 78 + 15, w = 56) for bromeliads occupied
by >1 metamorphosed anurans. The number of mature
leaves per A. zebrina was 14 to 46 (x = 28 + 6, w = 160),
and 17 to 43 (x = 29 + 6, w = 56) for bromeliads occupied
Table 1. Amphibians and reptiles collected or observed within Aechmea zebrina bromeliads. For each species the
number observed, height range (bromeliad in tree), and mean height are provided. Only metamorphed anurans at time
of collection included.
Species
Number observed
Height range (m)
Mean height (m)
Osteocephalus fuscifacies
3
24 . 3 - 28.1
25.6
Osteocephalus planiceps
1
31.5
-
Osteocephalus taurinus
1
30.6
-
Pristimantis acuminatus
1
40.4
-
Pristimantis aureolineatus
36
22 - 44.5
35.7
Pristimantis orphnolaimus
2
31 . 5 - 38.3
34.9
Pristimantis waoranii
35
21 . 2 - 43.9
31.9
Ranitomeya ventrimaculata
1
36.5
-
Ranitomeya variabilis
9
25 . 7 - 35.2
30.9
Scinax ruber
6
33 . 8-35
34.8
Anolis ortonii
1
20
-
Anolis transversalis
2
27-35
31
Thecadactylus solimoensis
1
31.5
-
Leptodeira annulata
1
43.5
-
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October 2014 I Volume 8 I Number 1 I e83
McCracken and Forstner
by >1 metamorphosed anurans. The water volume of A.
zebrina bromeliads was 42 to 3645 mL (x = 1343 + 656,
n = 160), and 355 to 3645 mL (x = 1428 + 726, n = 56)
for bromeliads oecupied by >1 metamorphosed anurans.
Water temperature within an outer leaf axil of A. zebrina
bromeliads at time of collection was 22.3 to 32.3 °C (x =
26.2 + 2.1, n = 160), and 22.6 to 31.2 °C (x = 26.2 + 1.9,
n = 56) for bromeliads occupied by >1 metamorphosed
anurans. Water pH within an outer leaf axil of A. zebrina
bromeliads at time of collection was 2.82 to 6.34 (x =
4.18 + 0.66, n = 160), and 3.22 to 6.34 (x = 4.34 + 0.73,
n = 56) for bromeliads occupied by >1 metamorphosed
anurans. Water pH of sieved homogenized water for each
A. zebrina bromeliad was 3.14 to 6.08 (x = 4.44 + 0.53,
n = 160), and 3.60 to 6.08 (x = 4.48 + 0.55, n = 56) for
bromeliads occupied by >1 metamorphosed anurans. Air
temperature adjacent to bromeliads at time of collection
was 21.1 to 34.6 °C (x = 27.8 + 2.8, n = 160), and 21.1 to
33.5 °C (x = 27.6 + 2.7, n = 56) for bromeliads occupied
by >1 metamorphosed anurans. Summary statistics for
bromeliads absent of anurans are contrasted with those
given above in Fig. 5.
By taking the mean number of metamorphosed an-
urans per bromeliad (x = 0.6) and multiplying by the
mean number of A. zebrina bromeliads per tree (x = 66),
we calculated an estimated mean of 39.6 metamorphosed
anurans occupying the A. zebrina bromeliads of an av-
erage tree in our study. The Dominant class tree crown
area from Asner et al. (2002) was 105.7 m^ (25.3 m tall)
with a calculated 37.5 anurans per 100 m^, and the Super
dominant class tree crown area was 311 m^ (46.1 m tall)
with a calculated 12.7 anurans per 100 m^.
Discussion
Our study identified 14 species of herpetofauna (10 an-
urans and four reptiles) utilizing the tank bromeliad A^c/^-
mea zebrina as habitat in the high canopy environment of
the northwestern Amazon Basin. A range of 1-5 meta-
morphosed anurans per bromeliad, with up to two spe-
cies occupying a single bromeliad, were detected in over
one-third of the bromeliads sampled. The observation of
larval, metamorphs, and adults of Osteocephalus fuscifa-
cies confirm that this species is a phytotelm breeder as
proposed by Jungfer et al. (2013). The observation of lar-
val, metamorphs, and an Sidult Ranitomeya ventrimacula-
ta confirm that this species does deposit tadpoles in high
canopy bromeliads as proposed by Brown et al. (2011).
Our detection of the gecko Thecadactylus solimoensis at
3 1 .5 m vertical height within the leaf axil of an A. zebrina
bromeliad confirms this species use of bromeliads in the
high canopy (Vitt and Zani 1997; Bergmann and Russell
2007). Our observation of the snake Leptodeira annulata
within the leaf axils of an A. zebrina bromeliad at 43.5
m in the canopy is the highest recorded vertical height to
our knowledge; L. annulata is described as terrestrial to
Amphib. Reptile Conserv. | amphibian-reptile-conservation.org (72)
semi-arboreal with a previous maximum observed ver-
tical height of 6 m above ground (Duellman 1978; Vitt
1996; Kacoliris 2006; Avila and Morals 2007).
In McCracken and Forstner (2014) we analyzed the
habitat data for differences among forest disturbance
treatments and found no differences in habitat variables
between treatments and no relationships between habitat
variables and anuran occupancy or abundance. Also, we
found differences between forest disturbance treatments
for anuran abundance and occupancy; but report the
summary statistics of the habitat data here as a resource
characterizing the habitat occupied by canopy tank bro-
meliad dwelling herpetofauna. Of particular interest in
this study was the mildly acidic mean water pH (4.18 in
situ in leaf axils, 4.34 in sieved homogenized water) in A.
zebrina bromeliads; as this is within the range reported
to affect development of embryonic and larval anurans
(Beattie and Tyler-Jones 1992). However, bromeliads are
a known breeding site for amphibians and we observed
an abundance of aquatic invertebrates and larval anurans
in our collections (Benzing 2000).
Using the two largest tree size classes of Amazonian
trees from Asner et al. (2002) as representative crown
area for the shortest (28 m) and tallest (49 m) trees in
our study, we calculated an estimate of 12.7-37.5 an-
urans per 100 m^ of crown area for an average tree in
our study. We consider this estimated range of canopy
anuran density to be conservative because 1) the height
of trees for the tree size classes used from Asner et al.
(2002) are shorter than our shortest and tallest trees; 2)
it is calculated on the two-dimensional space of the tree
crown and does not include the vertical space occupied
by a tree; 3) anurans were only collected from A. zebrina
bromeliads and not other available habitat; and 4) the
mean anuran abundance per tree in our study was used
for calculations of both tree size classes, not accounting
for the range of tree heights and number of bromeliads
per tree. Regardless of these constraints, the estimated
high anuran density of 37.5 anurans/100 m^ is the great-
est of any reported density for tropical frog assemblages
from comparable studies (e.g., 36.1 anurans/100 m2
at La Selva, Costa Rica [Lieberman 1986]; 15.5 an-
urans/100 m^ at Rio Llullapichis, Peru [Toft 1980]; see
also Allmon 1991 and Rocha et al. 2007 for compiled
sites comparison). The low estimate of 12.7 anurans/100
m^ is still amongst the highest densities of reported stud-
ies, particularly in South America (Allmon 1991; Rocha
et al. 2007). A limitation of this comparison is that these
studies rely on the method of quadrat surveys for density
calculations, where the majority of observed anurans are
going to be leaf-litter inhabitants or those that are within
arms reach (~2 m vertical height). Achieving a more ac-
curate canopy anuran density will require research sam-
pling all available canopy microhabitats and recording
crown measurements for all sampled trees.
Within the herpetological community the use of the
term “arboreal” has deviated from its recognized defini-
October 2014 I Volume 8 I Number 1 I e83
Herpetofaunal community of a high canopy tank bromeliad
tion of “inhabiting or frequenting trees” and taken on a
broader meaning in reference to vertical habitat use by am-
phibians and reptiles to simply mean living above ground
level (Merriam-Webster.com. 2014. Merriam-Webster
Dictionary. Available from http://www.merriam-webster.
com [Accessed 27 April 2014]). While this definition
suffices to distinguish these species (arboreal) from those
occupying fossorial and ground level habitat, it does not
adequately clarify the above ground vertical space uti-
lized by a particular species. As an example, Doan (2003)
reports the visual encounter survey (VES) method as the
best way to sample for arboreal herpetofauna in rainfor-
ests. The VES method only allows the researcher access
to habitat within arms reach (~2 m vertical height) and
fails entirely at observing animals within the other ~40-i-
m of vertical habitat above in many rainforests. Arboreal
herpetofauna may occur at vertical heights between >0 m
to 88 m on vegetation and/or trees; simply referring to a
species as arboreal provides no information about its oc-
cupied vertical range (Spickler et al. 2006). To alleviate
confusion and accurately represent the vertical space oc-
cupied by a species or assemblage we propose two alter-
natives to be used separately or preferably together. Eirst,
basic descriptors delineating vertical zones for a defined
forest type could be used to accompany “arboreal” (e.g.,
“arboreal within the understory” where “understory” has
been defined as “near-ground nondominant vegetation”)
(Dial et al. 2004). Second, authors should specify vertical
height ranges when describing or discussing “arboreal”
anurans (e.g., “the arboreal frog Pristimantis waoranii is
found in the overstory at 20.5^4 m” where “overstory”
has been defined as “high, dominant foliage”) (Dial et
al. 2004). Providing vertical range data or descriptions
is critical to understanding the many aspects of natural
history for a species.
Conclusion
The canopy of tropical forests are among the most
species-rich terrestrial habitats on Earth, yet remain a
relatively unexplored biotic frontier (Basset et al. 2003;
Lowman and Schowalter 2012). Our research has shown
the tank bromeliad Aechmea zebrina to support a di-
verse and abundant herpetofauna community in the harsh
equatorial tree canopy environment of the Yasum Bio-
sphere Reserve in the Amazon Basin of Ecuador. Addi-
tionally, our canopy work has contributed to the descrip-
tion of two new species of bromeliad-inhabiting anurans
{Pristimantis aureolineatus [Guayasamin et al. 2006]
and P. waoranii [McCracken et al. 2007]), the detection
of Batrachochytrium dendrobatidis (Chytrid fungus) on
anurans from the forest floor to the canopy in Amazo-
nia (McCracken et al. 2009), and identified the use of
high canopy bromeliads by the anuran Scinax ruber (Mc-
Cracken and Eorstner 2014). While canopy surveys of
tank bromeliads are labor intensive, they provide a very
Amphib. Reptile Conserv. | amphibian-reptile-conservation.org (73)
effective technique for collecting data on canopy inhabit-
ing organisms and associated microhabitat factors.
Our estimates of canopy anuran densities, based on
collections from a single species of bromeliad, demon-
strate the potential ecological importance and current
lack of knowledge on the canopy herpetofauna compo-
nent in tropical systems. Typical inventories of herpeto-
fauna in tropical forests are conducted at ground level (~2
m vertical height stratum) where microclimatic variables
are more stable (Guayasamin et al. 2006). Sampling such
shallow strata within the strongly vertical structure of
these forests has likely served to bias metrics of herpeto-
fauna assemblages by focusing on a narrow environmen-
tal space and neglecting the large available habitat above
into the canopy (Guayasamin et al. 2006; Scheffers et al.
2014). Euture inventory studies should routinely include
canopy surveys to properly represent the herpetofauna of
forested habitat.
Use of the term “arboreal” in the herpetofauna litera-
ture does not adequately deflne the vertical range of a
species or assemblage. This serves to limit compilation
and synthesis from the literature for the ecology of many
of these tropical reptiles and amphibians. Our proposed
amendments to accompany the description of arboreality
in herpetofauna functionally serve to give scale and pro-
vide a better understanding of the vertical habitat utilized
by a species or assemblage. As research on canopy her-
petofauna continues to expand, knowledge of the vertical
space occupied will be essential to answering hypothe-
sis-driven research questions and enacting sufficient con-
servation measures to protect all species.
Acknowledgments. — SEM sincerely thanks all the
fieldwork assistants who have contributed to this work
over the years. We thank all the staff at the Tiputini Bio-
diversity Station - Universidad San Erancisco de Quito
and the Yasuni Research Station - Pontificia Universi-
dad Catblica del Ecuador. We also thank the Waorani and
Kichwa peoples who allowed us to conduct fieldwork
in their territories. Thank you to Bejat McCracken for
everything, but especially the photography. Thank you
to Jerad Tullis in the Department of Geography at Texas
State University who constructed the mosaic satellite im-
age in Eigure 1. Lastly, we thank all our funding sources:
National Science Eoundation (Graduate Research Eel-
lowship Program and a GK-12 grant No. 0742306), Tex-
as State University - Department of Biology, the TAD-
POLE Organization, Sigma Xi - The Scientific Research
Society, Texas Academy of Science, and The Explorer’s
Club. This work was conducted under permit numbers
006-IC-EA-PNY-RSO and 012-IC-EA-PNY-RSO issued
by the Ministerio del Ambiente, Ecuador.
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Received: 01 May 2014
Accepted: 08 August 2014
Published: 30 October 2014
B Shawn F. McCracken is a Research Assistant Professor at Texas State University in San Marcos, Texas, USA.
He received his B.A. in biology and a Ph.D. in aquatic resources at Texas State University. He is the founder
and executive director of the TADPOLE Organization. His research interests include the conservation, ecol-
S9F ogy, and systematics of amphibians; with an emphasis on the effects of anthropogenic disturbance to amphib-
ian diversity and abundance in tropical rainforests. His current research focuses on tbe effects of deforestation
to canopy inhabiting herpetofauna and microclimate in Amazonian Ecuador, with a concentration on epiphytic
canopy tank bromeliads. In the USA, he conducts research on the endangered Houston toad (Anaxyrus hous-
tonensis) and the state threatened Texas tortoise (Gopherus berlandieri).
Michael R. J. Forstner is a Professor in Biology at Texas State University, and the Alexander-Stone Chair
in Genetics. He has a B.S. from Southwest Texas State University, M.S. from Sul Ross State University, and
a Ph.D. from Texas A&M University. He has broad interests in the effective conservation of rare taxa, par-
ticularly reptiles and amphibians. The students and colleagues working with him seek to provide genetic and
ecological data relevant to those conservation efforts.
Amphib. Reptile Conserv. | amphibian-reptile-conservation.org (75)
October 2014 I Volume 8 I Number 1 I e83
Copyright: © 2014 Torres-Carvajal et al. This is an open-access article distributed under the terms of the Creative
Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits unrestricted use
for non-commercial and education purposes only, in any medium, provided the original author and the official and
authorized publication sources are recognized and properly credited. The official and authorized publication credit
sources, which will be duly enforced, are as follows: official journal title Amphibian & Reptile Conservation-, of-
ficial journal website <amphibian-reptile-conservation.org>.
Amphibian & Reptiie Conservation
8(1) [Special Section]: 76-88.
A new species of Phoiidoboius (Squamata:
Gymnophthalmidae) from the Andes of southern Ecuador
^Omar Torres-Carvajal, ^Pablo J. Venegas, ^Simon E. Lobos, "^Paola Mafla-Endara,
and ^Pedro M. Sales Nunes
de Zoologia, Escuela de Ciencias Bioldgicas, Pontificia Universidad Catolica del Ecuador, Avenida 12 de Octubre 1076y Roca, Apartado
17-01-2184, Quito, ECUADOR ^Division de Herpetologia-Centro de Ornitologia y Biodiversidad (CORBIDI), Santa Rita N°105 36 Of. 202, Urb.
Huertos de San Antonio, Surco, Lima, PERILED epartamento de Ciencias Naturales, Universidad Tecnica Particular de Loja, San Cayetano Alto s/n
C.P 11 01 608, Loja, ECUADOR ^Universidade Federal de Pernambuco, Centro de Ciencias Bioldgicas, Departamento de Zoologia, Av. Professor
Moraes Rego, s/n. Cidade Universitdria CEP 50670-901, Recife, PE, BRAZIL
Abstract . — ^We describe a new species of Phoiidoboius lizard from the Amazonian slopes of the
Andes of southern Ecuador. Among other characters, the new species differs from other species of
Phoiidoboius in having a distinct diagonal white stripe extending from the fourth genial scale to the
fore limb. We present a phylogeny based on mitochondrial DNA sequence data as additional evidence
supporting delimitation of the new species, which is sister to all other species of Phoiidoboius.
Our phylogeny further supports the south-to-north speciation hypothesis proposed for other lizard
clades from the northern Andes.
Key words. Clade Phoiidoboius, DNA, lizard, phylogeny. South America, systematics
Citation: Torres-Carvajal O, Venegas PJ, Lobos SE, Mafla-Endara P, Nunes PMS. 2014. A new species of Phoiidoboius (Squamata: Gymnophthalmidae)
from the Andes of southern Ecuador. Amphibian & Reptile Conservation 8(1) [Special Section]: 76-88 (e84).
Introduction
The gynmophthalmid lizard clade Phoiidoboius was
recently defined by Torres-Carvajal and Mafla-Endara
(2013) as the largest crown clade conimnmg Phoiidoboius
montium Peters, 1863, but not Macropholidus ruthveni
Noble, 1921. This phylogenetic definition (de Queiroz
and Gauthier 1994) is based on a phylogenetic tree ob-
tained from analyses of mitochondrial DNA nucleotide
sequence data (Torres-Carvajal and Mafla-Endara 2013),
and is in conflict with previous non-phylogenetic defini-
tions of both Phoiidoboius and Macropholidus (Monta-
nucci 1973; Reeder 1996) based on morphological data.
As defined by Torres-Carvajal and Mafla-Endara (2013),
Phoiidoboius contains four species — P. affinis, P mac-
brydei, P montium, and P. prefrontalis. Contrary to pre-
vious taxonomic arrangements (Montanucci 1973; Reed-
er 1996), “P.” annectens was shown to be part of the
clade (traditionally ranked as a genus) Macropholidus.
In addition, Torres-Carvajal and Mafla-Endara (2013)
concluded that the controversial generic allocation of P.
anomalus from southern Peru (Montanucci 1973; Reeder
1996) still remains to be established.
Phoiidoboius lizards occur between 1,800 and 4,100
m along the southern part of the northern Andes (i.e.,
Ecuador and southern Colombia). Only one species, P.
macbrydei, occurs also in the Huancabamba Depression
in extreme southern Ecuador and possibly northern Peru.
Herein, we describe a new species of Phoiidoboius from
the Andes in southern Ecuador using data on morphology
and color pattern. We also present molecular evidence
supporting recognition of the new species by performing
phylogenetic analyses of nucleotide sequence data.
Methods
Morphological data: Type specimens and additional
specimens examined (Appendix 1) were deposited in the
herpetological collection at Museo de Zoologia, Ponti-
ficia Universidad Catolica del Ecuador, Quito (QCAZ).
The following measurements were taken with a digital
caliper and recorded to the nearest 0.1 mm, except for
tail length, which was taken with a ruler and recorded
to the nearest millimeter: head length (HE), head width
(HW), shank length (ShL), axilla-groin distance (AGD),
snout-vent length (SVL), and tail length (TL). Sex was
determined by dissection or by noting the presence of
everted hemipenes. We follow the terminology of Reeder
(1996) for description of the holotype and scale counts.
Data for other species of Phoiidoboius were taken from
Montanucci (1973).
The left hemipenis of two type specimens of the new
species (QCAZ 4998 and 4999) were prepared following
the procedures of Manzani and Abe (1988), as modified
by Pesantes (1994) and Zaher (1999), where the retractor
Correspondence. ^ omartorcar@gmail.com (Corresponding author); ^sancarranca@yahoo.es ; ^lobossimon@gmail.com;
"^paola. mmafen@gmail. com; ^pedro. nunes@gmail. com
Amphib. Reptile Conserv. | amphibian-reptile-conservation.org (76) November 2014 | Volume 8 | Number 1 | e84
Torres-Carvajal et al.
Fig. 1. Holotype (QCAZ 4998; SVL = 45.52 mm) of Pholidobolus hillisi sp. nov. in dorsal (A) and ventral (B) views. Photographs
by OTC.
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November 2014 I Volume 8 I Number 1 I e84
A new Pholidobolus from Ecuador
muscle is manually separated and the everted organ is
filled with stained petroleum jelly and paraffin. In addi-
tion, the hemipenial calcareous structures were stained
in an alcoholic solution of Alizarin Red, following the
adaptation of the procedures of Uzzell (1973) proposed
by Nunes et al. (2012). Description of the hemipenes fol-
lows the terminology of Dowling and Savage (1960),
Savage (1997), Myers and Donnelly (2001, 2008), and
Nunes et al. (2012).
DNA sequence data: Total genomic DNA was di-
gested and extracted from liver or muscle tissue using
a guanidinium isothiocyanate extraction protocol. Tissue
samples were first mixed with Proteinase K and a lysis
buffer and digested overnight prior to extraction. DNA
samples were quantified using a Nanodrop® ND-1000
(NanoDrop Technologies, Inc), re- suspended and diluted
to 25 ng/ul in ddH20 prior to amplification.
Using primers and amplification protocols from the
literature (Pellegrino et al. 2001; Torres-Carvajal and
Mafla-Endara 2013) we obtained 1,573 nucleotides (nt)
representing mitochondrial genes 12S (344 nt), 16S (549
nt), and ND4 (680 nt) from three individuals of the new
species described herein (GenBank accession numbers
KP090167-KP090175).
Chronophylogenetic analyses: We added the three
sequences generated in this study to the mtDNA dataset
of Torres-Carvajal and Mafla-Endara (2013). Editing,
assembly, and alignment of sequences were performed
with Geneious ProTM 5.3 (Biomatters Ltd. 2010). Genes
were combined into a single dataset with three partitions,
one per gene. The model of evolution for each partition
was obtained in jModeltest 2 (Darriba et al. 2012) under
the Akaike information criterion. Chronophylogenetic
analyses were performed in Beast 2.1.3 (Bouckaert et al.
2014) as described in Torres-Carvajal and Mafla-Endara
(2013), except that we performed four independent 108
generation runs with random starting trees, sampling ev-
ery 10,000 generations. The resultant 36,000 trees were
used to calculate posterior probabilities (PP) for each bi-
partition in a maximum clade credibility tree in TreeAn-
notator 2.1.2 (Rambaut and Drummond 2014).
Systematics: The taxonomic conclusions of this study
are based on the observation of morphological features
and color pattern, as well as inferred phylogenetic rela-
tionships. We consider this information as species de-
limitation criteria following a general lineage or unified
species concept (de Queiroz 1998, 2007).
Pholidobolus hillisi sp. nov.
urn:lsid:zoobank.org:act:EB5A9DDD-742C-456F-B5C9-6E57EDEEE698
Proposed standard English name: Cuilanes of Hillis
Proposed standard Spanish name: Cuilanes de Hillis
Amphib. Reptile Conserv. | amphibian-reptile-conservation.org (78)
Holotype: QCAZ 4998 (Figs. 1, 2), adult male, Ecua-
dor, Provincia Zamora-Chinchipe, near San Francisco
Research Station on Loja-Zamora road, 3°57’57”S,
79°4’45”W, WGS84, 1,840 m,21 July 2012, collected by
Santiago R. Ron, Andres Merino, Fernando Ayala, Teresa
Camacho, and Martin Cohen.
Paratypes (5): ECUADOR: Provincia Zamora-
Chinchipe: QCAZ 4999 (adult male), 5000 (juvenile
female), same data as holotype; QCAZ 6840 (adult fe-
male), 6842 (adult female), 6844 (adult male), San Fran-
cisco Research Station, 3°58’14”S, 79°4’41”W, WGS84,
1,840 m, 29 October 2004, 9 June 2005, and 29 Septem-
ber 2005, respectively, collected by Kristin Roos, Alban
Pfeiffer, Andy Fries, Ulf Soltau, and Florian Werner.
Diagnosis: Pholidobolus hillisi is unique among spe-
cies of Pholidobolus in having a distinct diagonal white
stripe on each side of the chin, extending from the fourth
genial to the fore limb (Fig. 3). It further differs from all
species of Pholidobolus, except P. affinis, in having three
supraoculars (two in P. macbrydei, P montium, and P
prefrontalis). Pholidobolus affinis differs from the new
species by having flanks with black reticulations on a
reddish orange ground color (flanks brown in P. hillisi'.
Fig. 4).
The new species also can be distinguished from P.
montium and P. macbrydei by the presence of prefrontal
scales (absent in the last two species). While P. hillisi
shares with P. affinis and P. prefrontalis the presence of
prefrontal scales, it differs from them in having a dark
brown dorsum with a conspicuous light brown vertebral
stripe (dorsum pale brown without a vertebral stripe in P.
affinis and P. prefrontalis'. Fig. 4). Furthermore, P. hillisi
has fewer dorsal scales in transverse rows (28-31) than
P. affinis (45-55), P. montium (35-50), P. prefrontalis
(37^6), and P. macbrydei (31^3).
Pholidobolus hillisi shares with all other recognized
species of Pholidobolus the absence of a single trans-
parent palpebral disc and the presence of a ventrolateral
fold between fore and hind limbs. These characters dis-
tinguish members of Pholidobolus from members of its
sister clade Macropholidus (Torres-Carvajal and Mafla-
Endara 2013).
Characterization: (1) Three supraoculars, anterior-
most larger than posterior one; (2) prefrontals present;
(3) femoral pores present in both sexes; (4) two to five
opaque lower eyelid scales; (5) scales on dorsal surface
of neck striated, becoming keeled from fore limbs to tail;
(6) two or four rows of lateral granules at midbody; (7)
28-31 dorsal scales between occipital and posterior mar-
gin of hind limb; (8) lateral body fold present; (9) keeled
ventrolateral scales on each side absent; (10) dorsum
dark brown with a conspicuous narrow, pale brown, ver-
tebral stripe that becomes grayish brown towards the tail;
(11) labial stripe white; (12) sides of body dark brown;
November 2014 I Volume 8 I Number 1 I e84
Torres-Carvajal et al.
Fig. 2. Head of the holotype (QCAZ 4998) of Pholidobolus hillisi sp. nov. in dorsal (A), lateral (B), and ventral (C) views. Photo-
graphs by OTC.
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November 2014 I Volume 8 I Number 1 I e84
A new Pholidobolus from Ecuador
Fig. 3. Head of five speeies of Pholidobolus in ventral view. (A) P. affinis', (B) P. hillisi sp. nov.; (C) P. macbryder, (D) P montiunr,
(E) P prefrontalis. Photographs by OTC.
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November 2014 I Volume 8 I Number 1 I e84
Torres-Carvajal et al.
(13) white stripe along fore limb present; (14) a distinct
diagonal white stripe on each side of the chin, extending
from the fourth genial to the fore limb; (15) adult males
with red flecks and ocelli (black with white center) dorsal
to insertion of fore and hind lim bs.
Description of hoiotype: Adult male (QCAZ 4998);
snout- vent length 45.52 mm; tail length 104 mm; dorsal
and lateral head scales juxtaposed, finely wrinkled; ros-
tral hexagonal, 2.09 times as wide as high; frontonasal
pentagonal, wider than long, laterally in contact with na-
sal, smaller than frontal; prefrontals pentagonal, nearly
as wide as long, with medial suture, laterally in contact
with loreal and first superciliary; frontal hexagonal, lon-
ger than wide, slightly wider anteriorly, in contact with
the prefrontals and supraoculars I and II on each side;
frontoparietals pentagonal, longer than wide, with me-
dial suture, each in contact laterally with supraoculars II
and III; interparietal roughly hexagonal, lateral borders
parallel to each other; parietals slightly smaller than in-
terparietal, tetragonal and positioned anterolaterally to
interparietal, each in contact laterally with supraocular
III and dorsalmost postocular; postparietals three, medial
scale smaller than laterals; supralabials seven, fourth lon-
gest and below the center of eye; infralabials five, fourth
below the center of eye; temporals enlarged, irregularly
hexagonal, juxtaposed, smooth; two large supratemporal
scales, smooth; nasal divided, irregularly pentagonal,
longer than wide, in contact with rostral anteriorly, first
and second supralabials ventrally, frontonasal dorsally,
loreal posterodorsally and frenocular posteroventrally;
nostril on ventral aspect of nasal, directed lateroposteri-
orly, piercing nasal suture; loreal rectangular; frenocular
enlarged, in contact with nasal, separating loreal from
supralabials; supraoculars three, with the first being the
largest; four elongate superciliaries, first one enlarged,
in contact with loreal; palpebral disk divided into two
scales, pigmented; suboculars three, elongated and simi-
lar in size; three postoculars, medial one smaller than the
others; ear opening vertically oval, without denticulate
margins; tympanum recessed into a shallow auditory me-
atus; mental semicircular, wider than long; postmental
pentagonal, slightly wider than long, followed posteri-
orly by four pairs of genials, the anterior two in contact
medially and the posterior two separated by postgenials;
all genials in contact with infralabials; gulars imbricate,
smooth, widened in two longitudinal rows; gular fold
incomplete; posterior row of gulars (collar) with four
scales, the medial two distinctly widened.
Scales on nape similar in size to dorsals, except for
the anteriormost that are widened; scales on sides of neck
small and granular; dorsal scales elongated, imbricate,
arranged in transverse rows; scales on dorsal surface of
neck striated, becoming keeled from fore limbs to the
tail; number of dorsal scales between occipital and poste-
rior margin of hind limbs 28; dorsal scale rows in a trans-
verse line at midbody 30; one row of smooth, enlarged
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ventrolateral scales on each side; dorsals separated from
ventrals by three rows of small scales at the level of the
13th row of ventrals; lateral body fold present; ventrals
smooth, wider than long, arranged in 20 transverse rows
between the collar fold and preanals; six ventral scales in
a transverse row at midbody; subcaudals smooth; limbs
overlap when adpressed against body; axillary region
composed of granular scales; scales on dorsal surface of
fore limb striated, imbricate; scales on ventral surface of
fore limb granular; two thick, smooth thenar scales; su-
pradigitals (left/right) 3/3 on finger I, 6/6 on II, 8/8 on III,
9/9 on IV, 6/6 on V; supradigitals 3/3 on toe I, 6/6 on II,
9/9 on III, 11/12 on IV, 8/8 on V; subdigital lamellae of
fore limb single, 5/5 on finger I, 8/9 on II, 13/13 on III,
14/14 on IV, 8/9 on V; subdigital lamellae on toes I and
II single, on toe III paired on the distal half, on toe IV all
paired, on toe V paired at the base; number of subdigi-
tal lamellae (pairs when applicable) 6/5 on toe I, 9/9 on
II, 13/14 on III, 19/20 on IV, 12/12 on V; groin region
with small, imbricate scales; scales on dorsal surface of
hind limbs striated and imbricated; scales on ventral sur-
face of hind limbs smooth; scales on posterior surface
of hind limbs granular; six femoral pores on each leg;
preanal pores absent; cloacal plate paired, bordered by
four scales anteriorly, of which the two medialmost are
enlarged.
Measurements (mm) and proportions of the hoiotype:
HL 12.6; HW 9.3; ShL 5.2; AGD 24.6; TL/SVL 1.72;
HL/SVL0.25; HW/SVL0.18; ShL/SVLO.lO; AGD/SVL
0.48.
Hemipenial morphology (Fig. 5): Both organs ex-
tend along approximately nine millimeters in length. The
lobes of the organs are fully everted and each hemipenis
is fully expanded.
The hemipenial body is roughly conical in shape,
with the base distinctly thinner than the rest of the organ,
ending in two small lobes with apical folds in the apex.
The sulcus spermaticus is central in position, originat-
ing at the base of the organ, which bears a fleshy fold
partially overlapping the sulcus spermaticus. From this
point on, the sulcus proceeds in a straight line towards
the lobes, and acquires an S-shape at the first third of the
body. The sulcus becomes broader at halfway the length
of the hemipenial body, and returns to its regular width
at the apical region; it gets divided in two branches at the
lobular crotch. Just before the crotch, the central region
of the sulcus bears a tiny fleshy fold, which is not part of
the sulcus division. From this point on, the two branch-
es of the sulcus run on the medial regions of the lobes
among conspicuous lobular folds. The sulcate face of the
hemipenial body presents two nude areas, parallel to the
sulcus spermaticus, which run throughout the hemipenial
body, getting thinner and encircling the base of the lobes.
The lateral and asulcate faces of the hemipenial body
are ornamented with 28-30 rows of roughly equidistant
flounces with calcareous spinules. The first four rows are
November 2014 I Volume 8 I Number 1 I e84
A new Pholidobolus from Ecuador
Fig. 4. Five species of Pholidobolus from Ecuador. (A) P. affinis',
sp. nov. Photographs by OTC (A, B, C, D) and S. R. Ron (E).
straight, with a large series of spinules on the central as-
pect of the asulcate face, and small isolated series of 5-6
spinules bordering the nude areas parallel to the sulcus
spermaticus. A V-shaped nude area at the central asulcate
face of the body separates the remaining flounces. The
flfth and sixth flounces are also interrupted laterally by
an extension of the basal nude area. From the seventh to
the apical-most one, the flounces cross the lateral aspects
of the organ from the sulcate to the asulcate face, initially
in roughly straight lines, gradually assuming chevron-
shapes and getting reduced in length towards the apex
of the organ.
The region between the asulcate and the lateral sur-
faces is marked by a conspicuous unevenness forming a
bulge, which is shared by closely related species, such as
Macropholidus annectens, M. huancabambae, M. ruth-
(B) P. macbrydei; (C) P montiunr, (D) P prefrontalis; (E) P hillisi
veni, Pholidobolus affinis, P macbrydei, P montium, and
P prefrontalis (Nunes, 2011).
Color of holotype in preservative: Dorsal back-
ground uniformly dark brown with a narrow light brown
vertebral stripe extending from occiput onto tail; ver-
tebral stripe slightly wider anteriorly; dorsal surface of
head light brown medially (rostral, frontonasal, prefron-
tals, frontal and frontoparietals) and dark brown laterally
(including supraoculars); white supralabial longitudinal
stripe extending from first supralabial to fore limb; lateral
aspect of neck dark brown with a dorsolateral light brown
stripe that extends posteriorly along the flanks to the hind
limbs; ventrolateral aspect of head and neck with a lon-
gitudinal white stripe extending posteriorly from fourth
genial to insertion of fore limb and then laterally along
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Torres-Carvajal et al.
Fig. 5. Left hemipenis of Pholidobolus hillisi sp. nov. (QCAZ 4999) in sulcate (left), lateral (middle), and asulcate (right) views.
Photographs by P Nunes.
upper arm; fore limbs with scattered ocelli (black with
white center); flanks grayish brown with two dorsolateral
stripes, the dorsal one light brown and the ventral one
dark brown; tail light brown dorsally and dark brown on
the sides; two and three well-defined, small ocelli (black
with white center) dorsal to insertion of fore and hind
limbs, respectively; ventral surface of head gray, with
dirty cream genials and scattered brown marks; chest,
belly and ventral surface of limbs and tail dark gray.
Variation: Measurements and scale counts of Pholi-
dobolus hillisi are presented in Table 1. Superciliaries
usually four, five in QCAZ 6840; supralabials usually
seven (eight of left side of specimen QCAZ 6840). Rows
of lateral granules at midbody two (QCAZ 4999, 6844) to
four (QCAZ 6842). Three specimens including the holo-
type, with a ventrolateral row of smooth enlarged scales
(QCAZ 4999, 6840). Specimen QCAZ 6842 has a tiny
scale separating the cloacal scales posteriorly; all four
scales bordering the cloacal plate anteriorly are similar
in size in two specimens (QCAZ 4999, 6844), whereas
the lateralmost scales overlap the cloacal scales in one
specimen (QCAZ 6840).
No variation was observed in color pattern in preser-
vative among adult males. They can be distinguished
from females by the presence of ocelli and pale flecks
around insertion of fore and hind limbs. Moreover, the
characteristic diagonal white stripe on each side of the
chin that extends from the fourth genial to the forearm is
more conspicuous in males than in females. Females are
larger (maximum SVL 55.7 mm, n=3) than males (maxi-
mum SVL 51.1 mm, n=3).
Coloration in life of an adult male paratype (QCAZ
4999) was similar to the holotype’s coloration in pre-
servative described above, except that specimen QCAZ
4999 had small red flecks both at insertion of fore limbs
Table 1. Sexual variation in lepidosis and measurements of Pholidobolus hillisi sp. nov. Range followed by mean + standard devia-
tion are given.
Character
Males (n=3)
Females (n=3)
Dorsal scales between occipital and posterior margin of hind limb
28-30 (29+1)
29-31 (30+1)
Dorsal scale rows in a transverse line at midbody
27-34 (30.33+3.51)
29-35 (31+3.46)
Ventral scales between collar fold and preanals
18-20 (20.33+1.15)
18-19 (18.67+0.58)
Ventral scale rows in a transverse line at midbody
6-7 (6.67+0.58)
6
Subdigital lamellae on Finger IV
14-15 (14.33+43.0)
13-15 (13.67+1.15)
Subdigital lamellae on Toe IV
19-20 (19.33+0.58)
19
Femoral pores
5-8 (6.33+1.52)
2-5 (3.5) (n=2)
Maximum SVL
51.1
55.7
TL/SVL
1.86 (n=l)
1.84-2.14(1.99) (n=2)
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A new Pholidobolus from Ecuador
0.15 0.10 0.05 0
Neusticurus rudls
Placosoma glabellum
Bachia flavescens
Cercosaura quadrilineata
Potamites ecpieopus
Proctoporus bolivianus
Riama cashcaensis
Macropholidus ruthveni
M. annectens
M. annectens
M. huancabambae
M. huancabambae
M. huancabambae
Pholidobolus hillisi sp. nov.
P hillisi sp. nov.
P. hillisi sp. nov.
P macbrydei
P. macbrydei
P. pre frontalis
P pre frontalis
P. montium
P montium
P affinis
P. affinis
Fig. 6. Maximum clade credibility tree inferred from the analysis of a dataset eontaining three mitochondrial genes under uneor-
related, log normally distributed rates; branch lengths are in substitutions per site. Posterior probability values are shown above
branches; asterisks correspond to values of 1.
extending onto sides of neck and at insertion of hind
limbs extending onto base of tail. In addition, the lateral
white stripe that starts on first supralabial extends further
posteriorly along flanks in specimen QCAZ 4999 (Fig.
4).
Phylogenetic relationships: The maximum clade
credibility tree resulting from the chronophylogenetic
analysis supports inclusion of the new species within the
Pholidobolus clade (Torres-Carvajal and Mafla-Endara
2013) with strong support (PP = 0.96; Fig. 6). Phyloge-
netic relationships among other species of Pholidobolus
and species of Macropholidus are identical to those ob-
tained by Torres-Carvajal and Mafla-Endara (2013).
Macropholidus ruthveni is sister (PP = 0.99) to a clade
containing both M. annectens and M. huancabambae (PP
= 1). Pholidobolus macbrydei is sister (PP = 0.91) to a
clade with the three remaining species of Pholidobolus;
the latter clade included P prefrontalis as sister (PP =
0.99) to a clade containing P. affinis and P. montium as
sister taxa (PP = 0.99). In contrast to the results reported
by Torres-Carvajal and Mafla-Endara (2013), the chrono-
phylogenetic tree inferred in this paper suggests that the
diversification of the clades Macropholidus and Pholi-
dobolus occurred at about the same time (Fig. 6).
Distribution and ecology: Pholidobolus hillisi inhab-
its low montane forests in the eastern slopes of the Andes
of southern Ecuador. This area represents a weather di-
vide between the humid Amazon and the dry Inter- Ande-
an regions (Beck et al. 2008). The new species is known
from Provincia Zamora-Chinchipe, at 1,840 m (Fig. 7),
in the deep valley of the Zamora river. The only gym-
71- w »-V¥ n-w
ii-W w Jl- w ta-ff Tf-'^
Fig. 7. Distribution of Pholidobolus in Ecuador. P. affinis (white
circles); P. macbrydei (blue circles); P. montium (green circles);
P. prefrontalis (orange circles); P hillisi sp. nov (red circle).
nophthalmid species known to occur in sympatry with
P. hillisi is Alopoglossus buckleyi, although P macbry-
dei is parapatrically distributed (Fig. 7). Two specimens
(QCAZ 4998, 4999) were found under logs and rocks
next to the Zamora river between 1 130 hrs and 1145 hrs.
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November 2014 I Volume 8 I Number 1 I e84
Torres-Carvajal et al.
whereas another specimen (QCAZ 5000) was basking
on a rock next to the road at 1200 hrs. Other specimens
(QCAZ 6840, 6842, 6844) were found and captured by a
domestic cat around the San Francisco Research Station
in pasture with interspersed shrubs.
Etymology: The specific epithet hillisi is a noun in the
genitive case and is a patronym for David M. Hillis, who
has had a great impact in the development of the field of
molecular systematics (e.g., Hillis et al. 1996). In par-
ticular, he published a classic paper on evolutionary ge-
netics of Pholidobolus lizards, where he compared some
phylogenetic tree reconstruction techniques and empha-
sized the importance of phylogenetics in biogeography
(Hillis 1985).
Remarks: The Andes of southern Ecuador and northern
Peru between 4°S and 7°S consist of relatively low-ele-
vation mountains that create a mixture of environments.
This region, known as the Huancabamba Depression, has
long been recognized as a major biogeographic barrier
for Andean organisms (e.g., Cadle 1991; Duellman 1979;
Vuilleumier 1969). Although all species of Pholidobolus,
except P. macbrydei, are restricted to the southern part
of the northern Andes (i.e., Ecuador and southern Co-
lombia), the new species described herein occurs on the
northern limit of the Huancabamba Depression.
The Huancabamba Depression seems to have in-
fluenced the radiation of several Andean lizard clades,
such as Stenocercus, Riama, Macropholidus, and Pholi-
dobolus (Doan 2003; Torres-Carvajal 2007; Torres-Car-
vajal and Mafla-Endara 2013). Except for Macropholi-
dus, these clades have diversified along the northern
Andes, suggesting that common geological or climatic
events have influenced these radiations. The phyloge-
netic tree presented in this paper further supports the idea
of a south-to-north sequence of speciation events (Doan
2003; Torres-Carvajal 2007) which is congruent with the
recent south-to-north uplift of the northern Andes (Simp-
son 1979; Aleman and Ramos 2000).
Acknowledgments. — We thank Santiago R. Ron for
photographs and Andrea Varela for assembling some
of the figures. Special thanks to Tiffany Doan and an
anonymous reviewer for their valuable comments. OTC
received funds from Secretaria de Educacion Superior,
Ciencia, Tecnologia e Innovacion (SENESCYT). PMSN
is grateful to Funda9ao de Amparo a Pesquisa do Estado
de Sao Paulo (FAPESP) for financial support (Grant #
2012/00492-8). Specimens were collected under collec-
tion permit 001-11 IC-FAU-DNB/MA issued by Ministe-
rio de Ambiente del Ecuador.
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Received: 10 September 2014
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Published: 12 November 2014
Additional specimens examined
P/70//C/060/US aff/n/s.— ECUADOR: Provincia Chimborazo: Colta, r41’56”S, 78°46’25”W, 3,215 m, QCAZ 9899-01 ; Sicalpa, 1°42’18”S,
78°46’32”W, 3,212 m, QCAZ 11887. Provincia Cotopaxi: Cutuchi river, San Miguel de Salcedo, 1°2’9”S, 78°35’53”W, 2,640 m, QCAZ
9641 . Provincia Tungurahua: 6 km N Mocha to 400 m Panamerican Highway, 1°22’1”S, 78°39’16”W, 3,205 m, QCAZ 9895-97; Ambato
surroundings, 1°14’59,8”S, 78°37’33”W, QCAZ 9340-73, 9375-9443; Chamisa on road to Guadalupe, r21’44”S, 78°30’39”W, 2,348
m, QCAZ 7266; Cotalo on path to Mucubi Community, 1°25’46”S, 78°31’3”W, 2,626 m, QCAZ 9839; Patate, 1°18’42”S, 78°30’36”W,
2,199 m, QCAZ 9847-50; Poatug Hamlet, Aya Samana, 1°16’58”S, 78°29’29”W, 2,573 m, QCAZ 10005, 10008, 10011-13, 10016,
10018; Poatug Hamlet, Terremoto, 1°16’23”S, 78°29’29”W, 2,547 m QCAZ 9997-10000, 10002-10004; San Miguelito on path to Pil-
laro, ri3’12”S, 78°31’31”W, 2,689 m, QCAZ 9844; San Miguelito on path to Teran, 1°12’58”S, 78°31’42”W, 2,741 m, QCAZ 9843.
Pholidobolus macbrydei.— ECUADOR. Provincia Azuay: 10 km S Cutchil, 3°8’2”S, 78°48’47”W, 2,900 m, QCAZ 823-24;1.2 km E Qs-
orancho, 2°46’8”S, 78°38’10”W, 2,390 m, QCAZ 826; 6.2 km S Cutchil, 3°6’32”S, 78°48’4”W, 2,800 m, QCAZ 827; 20 km NE Cuenca,
2°51’0”S, 78°51’14”W, QCAZ 1359; 7 km Sigsig, 2°59’56”S, 78°48’25”W, 2,890 m, QCAZ 1537; 6 km S Qha, 3°29’49”S, 79°9’47”W,
QCAZ 3658; 20 km Cuenca-EI Cajas, 2°46’39”S, 79°10’12”W, 3,508 m, QCAZ 9932-34, 9936-38, 10020; Cochapamba, 2°47’50”S,
79°24’56”W, 3,548 m, QCAZ 10133-35; Cochapata, 3°25’47”S, 79°3’35”W, 3,074 m, QCAZ 12605-07; Cuenca, Cuenca-Azoguez
Panamerican Highway 2°53’43”S, 78°57’30”W, 2,486 m, QCAZ 6985; El Cajas National Park, path to PatuI Community, 2°44’28”S,
79°14’5”W, 4,092 m, QCAZ 8010-11; El Cajas National Park, PatuI river, 2°41’37”S, 79°13’56”W, 3,610 m, QCAZ 8893; El Cajas Na-
tional Park, Zhurcay river, 3°2’30”S, 79°12’56”W, 3,766 m, QCAZ 8900-01; El Cajas National Park, 2°42’21”S, 79°13’32”W, 3,600 m,
QCAZ 8946; El Capo, 2°46’43”S, 79°14’43”W, 4,100 m, QCAZ 4997; Giron, San Gregorio Community, Quinsacocha paramo, 3°6’22”S,
79°13’4”W, 3,242 m, QCAZ 8510-11; Giron, San Gregorio Community, Quinsacocha paramo, 3°2’30”S, 79°12’56”W, 3,766 m, QCAZ
8894-99, 8902-05, 8907; Giron, San Gregorio Community, Quinsacocha paramo, 3°2’30”S, 79°12’57”W, 3,766 m, QCAZ 8906; Guablid,
2°46’30”S, 78°4r51”W, 2,453 m, QCAZ 991 3-1 7, 9919-20, 9940-41; Gualaceo-Limon road, 2°56’53”S, 78°42’43”, 3,110 m, QCAZ 819-
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Torres-Carvajal et al.
22; Gualaceo-Limon road, 8.1 km O Azuay-Morona Santiago border, 2°57’50”S, 78°427”W, 3,140 m, QCAZ 825; Gualaceo, 2°52’56”S,
78°46’31”W, 2,298 m, QCAZ 9606; Gualaceo-Plan de Milagro road, 2°54’35”S, 78°44’4”W, 2,624 m, QCAZ 10875; Las Tres Cruces,
2°46’30”S, 79°14”53”W, QCAZ 4136; Maylas, Gualaceo-Macas road, 2°58’25”S, 78°41’41”W, 3,100 m, QCAZ 7269; Mazan Protected
Forest, 2°52’29”S, 79°7’26”W, 2,700 m, QCAZ 1296-97; Mazan Protected Forest, 2°52’31”S, 79°7’45”W, 3,189 m, QCAZ 8008, 8013;
Qna-La Paz road, 3°22’42”S, 79°11’20”W, 2,969 m, QCAZ 6031; Patacocha hill, 3°7’16”S, 79°3’54”W, 3,340 m, QCAZ 6144; Pucara,
Tres Chorreras, 3°12’49”S, 79°28’3”W, QCAZ 11038; Quinoas river, 3°5’14”S, 79°16’40”W, 3,200 m, QCAZ 1564-66; San Antonio,
2°51’40”S, 79°22’43”W, 2,943 m, QCAZ 9668; San Vicente-Cruz path, 2°47’43”S, 78°42’53”W, 3,044 m, QCAZ 11416-17, 11420;
Sigsig, 3°7’46”S, 78°48’14”W, 2,969 m, QCAZ 5605-08; Sigsig road, 3°3’17”S, 78°47’19”W, 2,574 m, QCAZ 9605; Tarqui, 3°0’57”S,
79°2’40”W, 2,627 m, QCAZ 8512. Provincia Cahar: Canar, 2°33’39”S, 78°55’51”W, QCAZ 9947; Culebrillas, 2°25’35”S, 78°52’12”W,
4,000 m, QCAZ 1349; Guallicanga ravine, 2°25’56”S, 78°54’8”W, 3,960 m, QCAZ 10048-49; Guallicanga river, 2°28’24”S, 78°58’22”W,
3,048 m, QCAZ 10051-52; Ingapirca, 2°32’43”S, 78°52’28”W, 3,400 m, QCAZ 1551; Juncal, 2°28’24”S, 78°58’22”W, 3,048 m, QCAZ
10050; Mazar Protected Forest, 2°32’48”S, 78°41’54”W, QCAZ 7376-84, 7883; Mazar Reserve, La Libertad, 2°32’45”S, 78°41’46”W,
2,842 m, QCAZ 10970-72. Provincia Chimborazo: Alao, 10 km Fluamboya, 1°52’22”S, 78°29’51”W, 3,200 m, QCAZ 1567-68; Atillo
Grande, Magdalena lake, 2°11’15”S, 78°30’25”W, 3,556 m, QCAZ 9214; Atillo Grande, Frutatian lake, 2°12’57”S, 78°30’5”W, 3,700 m,
QCAZ 9216-18; Culebrillas, Sangay National Park, 1°57’39”S, 78°25’55”W, 3,345 m, QCAZ 9612; Pungala, Eten Community, Timbo,
1°55’45”S, 78°32’14”W, 3,408 m, QCAZ 9616-21 ; Pungala, Melan Community, 1°52’30”S, 78°32’52”W, 3,564 m, QCAZ 9626-29, 9631;
Qzogoche, 2°22’7”S, 78°41’20”W, 4,040 m, QCAZ 6006-07; Shulata, 2°20’22”S, 78°50’36”W, 3,228 m, QCAZ 5597-9;. Provincia El
Qro: Guanazan, 3°26’24”S, 79°29’13”W, 2,638 m, QCAZ 7891, 7894. Provincia Loja: 17.1 km S Saraguro, 3°43’45”S, 79°15’53”W,
3,150 m, QCAZ 828; 26 km N Loja, Fluashapamba Native Forest, 3°39’30”S, 79°16’20”W, 2,894 m, QCAZ 8651; Cordillera of Lagunil-
las, Jimbura, 4°49’1”S, 79°21’43”W, 3,600 m, QCAZ 3785; Cordillera of Lagunillas, Jimbura, 4°37’42”S, 79°27’49”W, 3,450 m, QCAZ
6145-47; Fierro Urco, 3°42’38”S, 79°18’18”W, 3,439 m, QCAZ 6949-50; Gurudel, 3°39’22”S, 79°9’47”W, 3,100 m, QCAZ 5078-79;
Jimbura, Jimbura lake, 4°42’32”S, 79°26’48”W, 3,036 m, QCAZ 6945-48; Jimbura, path to Jimbura lake, 4°42’34”S, 79°26’8”W, 3348
m, QCAZ 10054-62; Military antenna, Saraguro, 3°40’46”S, 79°14’16”W, 3,190 m, QCAZ 3673-75, 9632; San Lucas, 3°43’55”S,
79°15’38”W, 2,470 m, QCAZ 2861; Saraguro, 3°37’13”S, 79°14’9”W, 3,100 m, QCAZ 3606, 3754; Urdaneta, 3°36’6”S, 79°12’31”W,
QCAZ 201 9. Provincia Tungurahua: Poatug Flamlet, El Corral, 1°16’21”S, 78°28’5”W, 3,468 m, QCAZ 8047, 9995-96. Provincia Zamo-
ra Chinchipe: Loja-Podocarpus National Park road, 3°59’44”S, 79°8’28”W, 2,776 m, QCAZ 10870-71; Valladolid, Podocarpus National
Park, 4°29’3”S, 79°8’56”W, 1 ,800 m, QCAZ 3743.
Pholidobolus montium. — ECUADQR: Provincia Cotopaxi: 2 km S Chugchilan on road to Quilotoa, 0°48’24”S, 78°56’11”W, 2,917 m,
QCAZ 8056-58; Latacunga, 0°52’27”S, 78°38’26”W, 2,857 m, QCAZ 873-74, 1411-12, 9642; Mulalo, 0°46’35”S, 78°34’40”W, 3,030
m, QCAZ 9639; San Juan de Paste Calle, 0°45’4”S, 78°38’51”W, 1,956 m, QCAZ 8053-54; South llliniza, 0°39’43”S, 78°42’40”W,
3,400 m, QCAZ 858-59, 1454. Provincia Imbabura: Atuntaqui, 0°19’59”N, 78°12’50”W, QCAZ 855; Cotacahi, Peribuela, Cuicocha
Lake, Cotacachi-Cayapas Reserve, 0°17’34”N, 78°21’5”W, 3,082 m, QCAZ 9683, 9685-86; 0°23’4”N, 78°15’25”W, 2,900 m, QCAZ
6137, 6139; Cotacachi-Cayapas Reserve, Jose Marla Yerovi Islets, 0°18’20”N, 78°2T41”W, 3,093 m, QCAZ 10959-60; El Juncal,
0°26’6”N, 77°57’58”W, QCAZ 6451 . Provincia Pichincha: 16 km W Chillogallo, Quito-Chiriboga road, 0°17’46”S, 78°39’30”W, 3,100 m,
QCAZ 797; 5 km E Pifo-Papallacta road, 0°15’3”S, 78°17’58”W, 2,800 m, QCAZ 1107-08; Alambi, 0°1’59”S, 78°34’26”W, 2,727-3,800
m, QCAZ 9691; AlangasI, 0°18’24”S, 78°24’40”W, QCAZ 1453, 1469; Amaguaha, Hacienda San Ignacio, 0°22’22”S, 78°30’14”W,
QCAZ 1463-64, 5275; Calacall, Simon Bolivar Street, uphill through secondary road, 0°T1”N, 78°30’49”W, 3,001 m, QCAZ 11674,
11676, 11678-79; Calacall Stadium, 0°0’0,3”S, 78°30’38”W, 2,833 m, QCAZ 11682; Carretas, 0°6’25”S, 78°26’46”W, QCAZ 875;
Chillogallo, 0°16’48”S, 78°33’25”W, QCAZ 840-43; Cumbaya, La Primavera, 0°12’6”S, 78°25’40”W, QCAZ 7248; Guayllabamba,
0°3’23”S, 78°20’26”W, QCAZ 7905; Inga, 5.5 km SE La Merced, 0°17’51”S, 78°20’52”W, 2,798 m, QCAZ 5278; Lloa, 0°14’52”S,
78°34’33”W, QCAZ 4109; Lloa Stadium, 0°14’39”S, 78°35’12”W, 3,059 m, QCAZ 11661; Loreto, road to Molinuco, Central Stadium,
0°23’4”S, 78°24’30”W, 2,844 m, QCAZ 11663; Machachi, 0°29’50”S, 78°32’25”W, QCAZ 844-48, 1374-77, 1462; Machachi, The
Tesalia Springs Company S.A. surroundings, 0°30’27”S, 78°33’57”W, 2,900 m, QCAZ 1465-67, 830-31, 833, 860-61, 1459-61; None,
0°4’42”S, 78°34’24”W, 2,843 m, QCAZ 11653-55; None School, 0°4’4”S, 78°34’35”W, 2,754 m, QCAZ 11656-58; Pasochoa, 0°26’24”S,
78°30’15”W, 2,850 m, QCAZ 1451-52; Pomasqui, 0°3’3”S, 78°27’21”W, QCAZ 862-68; Pululahua Volcano, 0°2’34”N, 78°30’15”W,
QCAZ 1450, 1520; Quito, Bellavista, 0°11’21”S, 78°28’35”W, QCAZ 1099; Quito, Chillogallo, 0°16’26”S, 78°33’23”W, QCAZ 8967; Qui-
to, Itchimbla, 0°13’21”S, 78°29’56”W, QCAZ 834, 1455-58, 1643, 2843; Quito, Garden of the Pontificia Universidad Catolica del Ecua-
dor (PUCE), 0°12’33”S, 78°29’28”W, 2,800 m, QCAZ 856-57, 7032, 1295, 2853; Quito, Parque Metropolitano, 0°10’35”S, 78°27’40”W,
QCAZ 4051; Quito, Universidad Central del Ecuador, 0°1T59”S, 78°30’19”W, 2,800 m, QCAZ 3727; Rio Guajalito Protected Forest,
0°13’44”S, 78°48’22”W, QCAZ 1338-39; San Antonio de Pichincha, 0°0’33”S, 78°26’45”W, QCAZ 580-81 , 790-92, 849, 1119-20, 1368,
1393, 2220, 2223, 2653; Tababela, International Airport, 0°6’21”S, 78°21’4”W, QCAZ 8046, 9044, 10064, 10974-76; Quito, Tumbaco,
0°12’34”S, 78°24’2”W, QCAZ 1113-14; Uyumbicho, 0°22’59”S, 78°31’6”W, QCAZ 870.
Pholidobolus prefrontalis . — ECUADQR: Provincia Chimborazo: AlausI, 2°1T54”S, 78°50’42”W, 2359 m, QCAZ 9907-9911; Tixan,
2°9’22”S, 78°48’3”W, 2,908 m, QCAZ 9951-54.
Omar Torres-Carvajal graduated in Biological Sciences from Pontificia Universidad Catolica del Ecua-
dor (PUCE) in 1998, and in 2001 received a Master’s degree in Ecology and Evolutionary Biology from
the University of Kansas under the supervision of Dr. Einda Trueb. In 2005 he received a Ph.D. degree
from the same institution with the thesis entitled “Phylogenetic systematics of South American lizards
of the genus Stenocercus (Squamata: Iguania).” Between 2006-2008 he was a postdoctoral fellow at the
Smithsonian Institution, National Museum of Natural History, Washington DC, USA, working under the
supervision of Dr. Kevin de Queiroz. He is currently Curator of Reptiles at the Zoology Museum QCAZ of
PUCE and an Associate Professor at the Department of Biology in the same institution. He has published
more than 30 scientific papers on taxonomy, systematics, and biogeography of South American reptiles,
with emphasis on lizards. He is mainly interested in the theory and practice of phylogenetic systematics,
particularly as they relate to the evolutionary biology of lizards.
Amphib. Reptile Conserv. | amphibian-reptile-conservation.org (87)
November 2014 I Volume 8 I Number 1 I e84
A new Pholidobolus from Ecuador
Pablo J. Venegas graduated in Veterinary Medieine from Universidad Naeional Pedro Ruiz Gallo, Lam-
bayeque, Peru, in 2005. He is eurrently eurator of the herpetologieal eolleetion of Centro de Omitolo-
gia y Biodiversidad (CORBIDI) and researcher of the Museo de Zoologia QCAZ, Pontificia Universidad
Catolica del Ecuador in Quito. His current research interest is focused on the diversity and conservation
of the Neotropical herpetofauna with emphasis in Peru and Ecuador. So far he has published more than 30
scientific papers on taxonomy and systematics of Peruvian amphibians and reptiles.
Simon E. Lobos graduated in Biological Sciences from Pontificia Universidad Catolica del Ecuador
(PUCE) in 20 1 3 . As a student, he j oined the Museo de Zoologia QCAZ, Pontificia Universidad Catolica del
y Ecuador in Quito, where he developed a great interest in reptiles. He has been studying systematics of gym-
nophthalmid lizards for the last four years. For his undergraduate thesis, Simon worked on the “Molecular
systematics of lizard Alopoglossus (Autarchoglossa: Gymnophthalmidae) in Ecuador.” This manuscript is
1 the second lizard species description coauthored by Simon. Other papers based on his undergraduate thesis
® work are in preparation.
Paola Mafla-Endara graduated in Biological Sciences from Pontificia Universidad Catolica del Ecua-
dor (PUCE) in 2011. Her undergraduate thesis entitled “Phylogeography of Andean lizards Pholidobolus
(Squamata: Gymnophthalmidae) in Ecuador” provided her a gratifying knowledge about phylogenetics
systematics, evolution, statistics, and biogeography. Since this time, she has developed a deep interest in
molecular biology. Currently she works mostly in systematics and ecology of fungi. She is convinced that
the same knowledge can be useful to solve similar questions in different subjects. This manuscript repre-
sents the second lizard species description coauthored by Paola. Others are in preparation.
Pedro M. Sales Nunes graduated in Biological Sciences from Universidade de Sao Paulo (USP) in 2003,
and in 2006 received a Master’s degree in Zoology from the same institution under the supervision of
Dr. Hussam Zaher. In 2011 he received a Ph.D. degree from the same institution with the thesis entitled
“Hemipenial Morphology of the microteiid lizards (Squamata: Gymnophthalmidae)” under the supervi-
sion of Dr. Miguel Trefaut Rodrigues. Between 2012-2014 he was a postdoctoral fellow at the USP, Sao
Paulo, Brazil, also working under the supervision of Dr. Miguel Trefaut Rodrigues. He is currently Curator
of the Herpetologieal Collection at the Universidade Federal de Pernambuco (UFPE), Recife, Brazil, and
an Adjunct Professor at the Department of Zoology in the same institution. His production is focused on
taxonomy and systematics of South American reptiles, with emphasis in Squamata.
In accordance with the International Code ofZoologieal Nomenclature new rules and regulations (ICZN 2012), we have deposited this paper in publicly accessible institutional libraries.
The new species described herein has been registered in ZooBank (Polaszek 2005a, b), the official online registration system for the ICZN. The ZooBank publication LSID (Life Science
Identifier) for the new species described here can be viewed through any standard web browser by appending the LSID to the prefix “http://zoobank.org/”. The LSID for this publication
is: um:lsid:zoobank.org:pub:41593E9F-6F66-4E60-B073-2E8BF643358F.
Separate print-only edition of paper(s) (reprint) are available upon request as a print-on-demand service. Please inquire by sending a request to: Amphibian & Reptile Conservation
(amphibian-reptile-conservation.org; arc.publisher@gmail.com).
Amphibian & Reptile Conservation is a Content Partner with the Encyclopedia of Life (EOL); http:///www.eol.org/ and submits information about new species to the EOL freely.
Digital archiving of this paper are found at the following institutions: ZenScientist (http://www.zenscientist.com/index.php/filedrawer); Ernst Mayr Library, Museum of Comparative Zool-
ogy, Harvard University, Cambridge, Massachusetts (USA); Florida Museum of Natural History, Gainesville, Florida (USA).
Complete journal archiving is found at: ZenScientist (http://www.zenscientist.com/index.php/filedrawer); Florida Museum of Natural History, Gainesville, Florida (USA).
Citations
ICZN. 2012. Amendment of Articles 8,9,10,21 and 78 of the International Code of Zoological Nomenclature to expand and refine methods of publication. Zootaxa 3450: 1-7.
Polaszek A et al. 2005a. Commentary: A universal register for animal names. Nature 437: 477.
Polaszek A et al. 2005b. ZooBank: The open-access register for zoological taxonomy: Technical Discussion Paper. Bulletin of Zoological Nomenclature 62(4): 210-220.
Amphib. Reptile Conserv. | amphibian-reptile-conservation.org (88)
November 2014 I Volume 8 I Number 1 I e84
Official journal website:
amphibian-reptile-conservation.org
Amphibian & Reptiie Conservation
8(1) [Special Section]: 89-106 (e88).
Early development of the glass frogs Hyalinobatrachium
fleischmanni and Espadarana callistomma (Anura:
Centrolenidae) from cleavage to tadpole hatching
Man'a-Jose Salazar-Nicholls and Eugenia M. del Pino*
Escuela de Ciencias Biologicas, Pontificia Universidad Catolica del Ecuador, Av. 12 de Octubre 1076 y Roca, Quito 170517, ECUADOR
Abstract— Wfe report the characteristics of embryonic development from cleavage to tadpole
hatching in two species of glass frogs, Hyaiinobatrachium fieischmanni and Espadarana
caliistomma (Anura: Centrolenidae). This analysis of embryonic development in centrolenid frogs
enhances comparative studies of frog early development and contributes baseline information for
the conservation and management of Ecuadorian frogs. These frogs reproduced in captivity and
their embryos were fixed for developmental analysis. The morphology of embryos was evaluated
in whole mounts, bisections, thick sections, and fluorescent staining of cell nuclei. Egg clutches
contained an average of 23 and 35 eggs for H. fieischmanni and E. cailistomma, respectively. The
eggs of both frogs measured approximately 2.1 mm in diameter. The eggs of H. fieischmanni were
uniformly pale green. In contrast, the animal hemisphere of E. callistomma eggs was dark brown
and the vegetal hemisphere was light brown. The developmental time of H. fieischmanni and E.
caliistomma under laboratory conditions was 6 and 12 days, respectively from the 32-cell stage
until tadpole hatching. Differences in environmental conditions may be associated with the time
differences of early development observed in these frogs. The development of glass frogs from egg
deposition to tadpole hatching was staged into 25 standard stages according to the generalized
table of frog development. Archenteron elongation began in the early gastrula and notochord
elongation began in mid to late gastrula, as in X. laevis. Development of the gastrocoel roof plate
(grp) was precocious in the two centrolenid frogs. The grp was detected in the late gastrula of both
species; whereas the grp was detected in neurula stages of X. laevis. The presence of the grp in
embryos of these frogs suggests that the mechanisms of left-right asymmetry, found in X. iaevis and
other amphibians, may be shared by these centrolenid frogs. The development of H. fieischmanni
and E. caiiistomma resembles the pattern found in frogs with rapid development such as the
aquatic eggs of X. iaevis and the development in floating foam-nests in the genus Engystomops
(Leptodactylidae). Differences in egg pigmentation were particularly significant in connection with
the divergent reproductive strategies of these glass frogs.
Key words. Developmental time, egg pigmentation, embryonic development, gastmlation, gastrocoel roof plate, neu-
mla
Citation: Salazar-Nicholls M-J, del Pino EM. 2015. Early development of the glass frogs Hyalinobatrachium fieischmanni and Espadarana callis-
tomma (Anura: Centrolenidae) from cleavage to tadpole hatching. Amphibian & Reptile Conservation 8(1) [Special Section]; 89-106 (e88).
Copyright: © 2015 Salazar-Nicholls and del Pino. This is an open-access article distributed under the terms of the Creative Commons Attribution-
NonCommercial-NoDerivatives 4.0 International License, which permits unrestricted use for non-commercial and education purposes only, in any me-
dium, provided the original author and the official and authorized publication sources are recognized and properly credited. The official and authorized
publication credit sources, which will be duly enforced, are as follows: official journal title Amphibian & Reptile Conservation] official journal website
<amphibian-reptile-conservation. org> .
Received: 13 May 2014; Accepted: 19 December 2014; Published: 27 Feburary 2015
Correspondence. Email: *edelpino@puce.edu.ec (Corresponding author, Eugenia M. del Pino); tel: (593 2) 299 1 700 extension
1280; fax: (593 2) 299 1725.
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Salazar-Nicholls and del Pino
Introduction
Centrolenid frogs are commonly known as glass frogs
because the internal organs of the adult are visible
through the transparent ventral body wall. This transpar-
ent region varies in size among species (Fig. 1 A-B, D-E)
(Cisneros-Heredia and McDiarmid 2007). Glass frogs
are endemic to the tropical regions of South America
from Venezuela to northern Argentina and south-eastern
Brazil (AmphibiaWeb 2014) and are particularly diverse
in the cloud forests of Colombia and Ecuador (Delia et
al. 2010; Guayasamin and Tmeb 2007; Ospina-Sarria et
al. 2010). These arboreal frogs deposit their eggs in ge-
latinous masses on the upper or lower surface of plant
leaves bordering stream banks. After hatching, the tad-
poles drop into the underlying streams. Tadpoles are fos-
sorial and live in the substrate along the shoreline (Delia
et al. 2010; Duellman and Tmeb 1986).
We studied the early development of the glass frogs
Hyalinobatrachium fleischmanni and Espadarana cal-
listomma (Anura: Centrolenidae) to compare their de-
velopment with frogs that exemplify different reproduc-
tive modes and to contribute to the knowledge of frogs
from Ecuador. Development of these centrolenid frogs
was compared with the embryogenesis of Tungara frogs,
Engystomops (Leptodactylidae). Tungara frogs constmct
foam nests that float in the water (Romero-Carvajal et
al. 2009). In addition, this comparison was extended to
the terrestrial embryos of poison arrow frogs (Dendro-
batidae), embryos of the Marsupial frog, Gastrotheca
riobambae (Hemiphractidae), and the aquatic embryos
of Xenopus laevis (Pipidae) and Ceratophrys stolzman-
ni (Ceratophryidae) (Elinson and del Pino 2012; Nieu-
wkoop and Eaber 1994; del Pino et al. 2004) (Table 1).
The analysis of H. fleischmanni and E. callistomma early
development was feasible because of the recent success-
ful reproduction of centrolenid frogs in captivity at the
Balsa de los Sapos, Centre of Amphibian Investigation
and Conservation (CICA), Pontificia Universidad Catdli-
ca del Ecuador (PUCE).
Hyalinobatrachium fleischmanni (Fig. lA-C) occurs
from southern Mexico to northern South America, in-
cluding Ecuador. The egg clutches consist of 20-40 pale-
green eggs, attached to the underside of plant leaves (Eig.
1C). Parental care of the egg clutch is provided by the
male to maintain the needed humidity. The male prevents
predation by katydids, wasps, ants, and other insects by
kicking with his limbs at the predatory insect (Delia et al.
2010; Greer and Wells 1980; Savage 2002).
Espadarana callistomma (Guayasamin and Tmeb
2007) (Pig. ID-F) occurs in the lowlands of northeastern
Ecuador and southern Colombia (Guayasamin and Tmeb
2007; Ospina-Sarria et al. 2010). Darkly pigmented
eggs are deposited on the upper surface of plant leaves
(Guayasamin and Tmeb 2007) (Pig. IF). Egg predation
by insects has not been reported for this species.
The left-right asymmetric location of organs, such
as the liver and the heart is established in the X. laevis
gastrocoel roof plate (grp) of the neumla by fluid-flow
towards the left side, guided by the clockwise rotation
of cilia (Blum et al. 2009; Schweickert et al. 2010). The
rotation of cilia in the frog grp, or in equivalent stmctures
of other vertebrates, determines the asymmetric expres-
sion of the gene Nodal in the grp left side (Blum et al.
2014b). The grp of X. laevis derives from the superflcial
prospective mesoderm of the early gastmla that becomes
internalized during gastmlation, and ends up in the dorsal
roof of the primitive gut. The grp can be detected by the
presence of exposed mesoderm corresponding to the no-
tochord and some paraxial mesoderm in the dorsal roof
of the primitive gut, and it is bordered by the lateral en-
dodermal crests (lee). As development advances, the lee
close to the midline and the primitive gut cavity becomes
totally lined with endoderm (Blum et al. 2009). The left-
right asymmetry is determined by fluid flow guided by
cilia rotation in the grp of frogs and other vertebrates.
However, a comparable structure to the grp has not been
reported for the chick and pig, and the left-right symme-
try breakage in these vertebrates may depend on a modi-
fled mechanism (Blum et al. 2014a, b; Saenz-Ponce et
al. 2012b). We analyzed the presence of the grp in the
gastmla and neumla of glass frogs to provide additional
comparison.
We characterized the embryos of these glass frogs
from cleavage to hatching of the tadpoles. We found that
in glass frogs, gastmlation overlapped with body elonga-
tion, as in frogs with rapid embryonic development. The
grp was detected in the late gastmla of both species of
glass frogs. Its presence suggests that the mechanisms
of left-right asymmetry, found in X. laevis and other am-
phibians, may be shared by these centrolenid frogs. The
reproductive mode of these glass frogs is associated with
rapid development. The strategy of egg deposition in the
underside or upper surface of leaves is associated with
differences in developmental time and pigmentation of
embryos and tadpoles.
Materials and Methods
Locality of collection and staging of embryos. Hya-
linobatrachium fleischmanni and Espadarana callis-
tomma were collected from Esmeraldas Province, San
Lorenzo, Durango, along the banks of the Rio Durango
and its tributaries in northwest Ecuador. The altitude of
this site is 243 m above sea level, and the geographic co-
ordinates are W 78.62405, N 1.04186. Erogs of both spe-
cies were collected on 04 October 2009 by Elicio Tapia
and Santiago Garcia. The adults successfully reproduced
at the Balsa de los Sapos, Centre of Amphibian Investi-
gation and Conservation (CICA), School of Biological
Sciences, Pontiflcia Universidad Catolica del Ecuador
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Early development of Hyalinobatrachium fleischmanni and Espadarana callistomma
H. fleischmanni
E. callistomma
Fig. 1. The adults and egg clutches of glass frogs. (A-C) Hyalinobatrachium fleischmanni. (A) Lateral view of an adult male. (B)
Ventral view of an adult male. The arrow indicates the border of the transparent body wall. The intestine and a blood vessel are vis-
ible. (C) Partial view of an egg clutch at the gastrula stage. The embryos are uniformly pale and the blastocoel roof is translucent
(arrowhead). (D-F) Espadarana callistomma. (D) Lateral view of an adult female. (E) Ventral view of an adult female. The arrow
signals the pigmented oocytes visible through the transparent body wall. The size of the transparent region is smaller than in H.
fleischmanni shown in B. (F) Partial view of an egg clutch. The embryos were at stages 5-6 (Table 2). Photographs of adult frogs
by Santiago Ron (A—B, D—E).
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(PUCE). The permit 016-IC-FAU-DNBAP-MA from the
Ministry of the Environment, Ecuador, allowed the col-
lection and maintenance of these frogs at Balsa de Sapos.
Egg clutches were donated to the Laboratory of Develop-
mental Biology for analysis of embryonic development.
This study was based on the analysis of embryos derived
from seven egg clutches of H. fleischmanni and four egg
clutches of E. callistomma.
The number of eggs of each egg clutch was record-
ed and the embryos were cultured in humid chambers
at room temperature, as described for embryos of the
dendrobatid frog, E. machalilla (del Pino et al. 2004).
At various intervals, some embryos were moved to a
Petri dish filled with 15% Steinberg’s solution (del Pino
et al. 2004) and the egg-jelly was manually removed to
study embryogenesis. Embryos were staged according
to the general table of frog development (Gosner 1960).
Egg diameter was measured in fixed embryos with the
measuring tool of the program, Axiovision (Carl Zeiss,
Oberkochen, Germany).
Fixation, staining and analysis of embryonic devel-
opment. Embryos were fixed in Smith’s fixative (del
Pino et al. 2004). The procedures for the bisection of
embryos, vibratome sectioning, cell nuclei staining with
the fluorescent dye Hoechst 33258 (Sigma- Aldrich, St.
Louis, MO, USA), and the staining of cell boundaries
with silver nitrate were previously described (Moya et
al. 2007; del Pino et al. 2004). Sections were mounted
in glycerol, and were examined with a Stemi SV6 stereo
microscope (Carl Zeiss, Oberkochen, Germany) or with
fluorescent optics using a Z1 Axio Observer microscope
(Carl Zeiss, Oberkochen, Germany). Embryos were pho-
tographed with Axiocam cameras, attached to micro-
scopes, and the image capture program, Axiovision (Carl
Zeiss, Oberkochen, Germany). The images were edited
with Adobe Photoshop CS6.
Results and Discussion
Internal organs of adult glass frogs can be observed
through their transparent belly; however the size of the
transparent window varies in the different genera of cen-
trolenid frogs as detected for H. fleischmanni and E. cal-
listomma (Fig. lA-B, D-E) (Cisneros-Heredia and Mc-
diarmid 2007). In contrast with adults, the eggs of these
frogs were opaque (Fig. 1C, F). We also detected sig-
nificant pigmentation differences as the H. fleischmanni
eggs and embryos were pale-green and those of E. cal-
listomma were dark brown (Fig. 1C, F). Egg pigmenta-
tion is a distinctive character of the different genera of
Centrolenidae; moreover some species deposit their eggs
in the upperside and others in the underside of leaves.
However, some species show no particular preference
for the upper or underside of leaves for the deposition of
their eggs (Cisneros-Heredia and Mcdiarmid 2007).
Clutch size, egg pigmentation and developmental
time. The number of eggs ranged from 14-30 eggs, with
a mean of 23 eggs per clutch in H. fleischmanni, and 32-
39 eggs, with a mean of 35 eggs per clutch in E. callis-
tomma. The eggs of both species measured about 2.1 mm
in diameter (Table 1). The embryos of H. fleischmanni
were uniformly pale-green (Figs. 1C; 2A-L; 3A-D). In
contrast, the animal hemisphere of E. callistomma em-
bryos was dark brown, and the vegetal hemisphere was
pale-brown (Figs. IF; 4A-L; 5A-F).
Dark pigmentation of the animal hemisphere of the
egg may provide protection against solar UV radiation
and may capture solar heat required to accelerate early
development of frog embryos exposed to solar radiation
in moist or aquatic environments. In contrast, there is lack
of dark pigment in frog eggs and embryos that develop
in secluded places (Duellman and Tmeb 1986; Elinson
and del Pino 2012). We propose that H. fleischmanni
embryos do not require dark pigmentation because the
underside of plant leaves may provide protection against
solar radiation. In contrast, the presence of dark pigment
in eggs and embryos of E. callistomma may be needed,
as the egg clutches are directly exposed to UV solar ra-
diation on the upper surface of plant leaves.
The differences in pigmentation were detectable in
eggs and embryos until tadpole hatching (Figs. 1C, F;
2-5). At hatching, the tadpoles of H. fleischmanni were
pale green with little dark pigmentation on the dorsum;
whereas E. callistomma tadpoles had a brown color
(Figs. 3C-D; 5F). The fossorial free-living tadpoles of
H. fleischmanni remained nearly unpigmented had elon-
gated bodies, and narrow tail fins to enable digging in the
sandy stream bottoms. The eyes were reduced in size and
were covered by skin characters likely associated with
the fossorial habits of H. fleischmanni tadpoles (Delia et
al. 2010; Duellman and Tmeb 1986; Savage 2002).The
characteristics of the E. callistomma free-living tadpoles
are unknown. The differences in tadpole pigmentation at
hatching suggest that the larval stages of these two cen-
trolenids may occur in dissimilar aquatic environments.
The differences in egg pigmentation observed in H.
fleischmanni and E. callistomma may depend on differ-
ent expression levels of the gene Shroom2 during oogen-
esis. Shroom2, an actin-binding protein, controls pig-
ment granule localization in the animal cortex ofX. laevis
oocytes (Lee et al. 2009). The oocytes of Engystomops
pustulosus (Leptodactylidae) contain small amounts of
Shroom2 protein and are white in color. However, Engys-
tomops embryos have dark pigment granules around nu-
clei of blastomeres (Lee et al. 2009; Romero-Carvajal et
al. 2009). Embryos of H. fleischmanni are pale and do
not have dark pigment around the nuclei of blastomeres;
whereas, in E. callistomma embryos dark pigment was
observed on the cell surface of animal pole blastomeres,
as well as around blastomere nuclei.
Embryos of H. fleischmanni and E. callistomma were
maintained under identical laboratory conditions with a
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Hyalinobatrachium fleischmanni
Fig. 2. External morphology of Hyalinobatrachium fleischmanni embryos from cleavage to the tail bud stage. (A) Stage 7: Thirty-
two cell stage. Animal micromeres were much smaller than the vegetal macromeres. (B) Stage 8: Mid cleavage. (C) Stage 9:
Blastula. (D) Stage 10.5: Early gastrula. The dorsal blastopore lip can be seen in the dorsal subequatorial region. (E) Stage 11: Mid
gastrula. The yolk plug was large. (F) Stage 12: Late gastrula. (G) Stage 12.5: Late gastrula with a small yolk plug. Neural groove
and neural plate were visible in embryos of this stage. (H) Stage 12.75: Late gastrula. The neural groove was visible. The yolk
plug was small. (I) Stage 14: Early neural fold. (J) Stage 15: Mid-neural fold. The neural folds were more elevated. (K) Stage 16.
Closure of the neural tube. The neural folds were near each other. (L) Stage 17. Tail bud stage. The branchial arches were visible.
In this and the following figures, numbers in the top right-hand comer give the developmental stage, br, branchial arch; c, cleavage
furrow; dl, dorsal blastopore lip; hy, hyoid arch; ma, mandibular arch; nf, neural fold; ng, neural groove; np, neural plate; vl, ventral
blastopore lip; yp, yolk plug.
temperature fluctuation of 18-23 °C. However, develop-
mental time diverged greatly between these frogs, as em-
bryos of H. fleischmanni required six days and those of
E. callistomma required 12 days from the 32-cell stage
until tadpole hatching. However in nature, great variation
in developmental time was observed in H. fleischmanni,
as egg clutches required 8-21 days from oviposition to
tadpole hatching (Greer and Wells 1980). In our labora-
tory, development of H. fleischmanni and E. callistomma
was slower than in the floating foam-nests of Engysto-
mops (Leptodactylidae), and faster than in the terrestrial
nests of Dendrobatidae. In two species of Engystomops,
development from egg deposition until hatching required
only three days whereas 19-21 days were required for
the same developmental processes by six species of den-
drobatid frogs (del Pino et al. 2004, 2007; Romero-Car-
vajal et al. 2009) (Table 1).
Reproductive strategies. We propose that rapid devel-
opment may be favored in H. fleischmanni in compari-
son with E. callistomma because eggs deposited on the
underside of plant leaves are at a greater risk of desicca-
tion in comparison with eggs deposited on the upperside
of leaves (Delia et al. 2010; Savage 2002). Moreover,
rapid development may be required in all centrolenids,
including frogs of the genus Espadarana, to overcome
predation from a number of insect families and other
arthropods (Cabanzo-Olarte et al. 2013; Duellman and
Tmeb 1986; Villa 1977; Vockenhuber et al. 2008). The
deposition of eggs on the underside of plant leaves and
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Table 1. Comparison of reproductive and developmental characteristics of glass frogs.
Family and Species
Reproduction
Clutch size
and (egg
diameter,
mm)
Gastrulation
time (hrs)®
Presence of
the grp in
the neurula
Onset of noto-
chord elongation
Refs“
Rapid Development
1
Centrolenidae
Hyalinobatrachium fleischmanni
Leaves underside
23 (2.1)
24
Yes
mid gastrula'’
2
Espadarana callistomma
Leptodactylidae
Leaves upperside
35 (2.1)
23
Yes
mid gastrula'’
2
Engystomops randi
Floating foam-nest
110(1.1)
12.5
Yes
mid gastrula'’
3,4
Engystomops coloradorum
Floating foam-nest
130(1.3)
12.5
Unknown
mid gastrula'’
3
Ceratophryidae
Ceratophrys stolzmanni
Pipidae
Aquatic
664 (2.2)
5
Yes
mid gastrula'’
4,5
Xenopus laevis
Aquatic
1000(1.2)
6
Yes
mid gastrula'’
6,7
Slow Development
1
Dendrobatidae
Epipedobates machalilla
Terrestrial nest
15 (1.6)
65
Yes
After gastrulation^
4,8
Epipedobates tricolor
Hemiphractidae
Terrestrial nest
13 (2.0)
36
Yes
After gastrulation^
8,9
Gastrotheca riobambae
Egg brooding
128 (3.0)
168
Yes
After gastrulation^
1,4
'‘Time from stages 10-13. Embryo culture temperatures for: X laevis 23 °C, and 18-23 °C for other frogs.; '’Stage 11; ‘‘Stage 13;
‘'References: 1, (del Pino et al. 2007); 2, This work; 3, (Romero-Carvajal et al. 2009); 4, (Saenz-Ponce et al. 2012b); 5, (Ortiz, 2013);
6, (Nieuwkoop and Faber 1994); 7, (Blum, et al. 2009); 8, (del Pino et al. 2004); 9, (Saenz-Ponce et al. 2012a).
predation of eggs and embryos by wasps, ants, katydids
and other arthropods are likely determining factors in fa-
vor of rapid development in H. fleischmanni.
Aquatic eggs and embryos characterize the basal
mode of frog reproduction, as exemplified by X. laevis
and Ceratophrys stolzmanni (Table 1). These frogs re-
lease a large number of small eggs in the water. However,
frogs have invaded different environments for reproduc-
tion due to competition for water resources, predation,
and the dangers of desiccation. Accordingly, clutch size,
egg size and developmental time vary among species
(Table 1) (Duellman and Tmeb 1986). The dissimilar de-
velopmental times of H. fleischmanni and E. callistomma
may relate to their egg deposition sites and to different
predation pressure on eggs and embryos. Egg deposition
in the upperside or underside of leaves associated with
differences in egg pigmentation and developmental time,
as observed in centrolenid frogs, are different reproduc-
tive modes that deserve further investigation.
Development of H. fleischmanni and E. callistomma.
The characteristics of development are detailed in Table
2, and illustrated in Figs. 2-13. It was of interest to docu-
ment the characteristics of development of these glass
frogs, given the observed differences in embryonic pig-
mentation and developmental time. The development
from early cleavage to tadpole hatching of H. fleischman-
ni and E. callistomma was characterized according to the
generalized table of frog development (Gosner 1960)
(Table 2). Embryos of H. fleischmanni from fertilization
to the sixteen cell stage were not available.
Micrographs of the external morphology of embryos
illustrate the developmental stages of both species, and
clearly demonstrate the pigmentation differences among
species (Figs. 1C, F; 2A-F; 3A-D; 4A-F; 5A-F). The
internal morphology of embryos from cleavage until the
completion of neurulation follows the typical frog pat-
tern, as outlined in the generalized table of development
(Gosner 1960) (Figs. 6-13). The most notable differenc-
es are the overlap between gastrulation and the onset of
neural development, and the lack of pigment in embryos
of H. fleischmanni in comparison with embryos of E. cal-
listomma. In both species cleavage was holoblastic (Figs.
6A-D; 7A-D), and the blastocoel roof was reduced to
two-cells in thickness during gastrulation. At gastrula-
tion, a conspicuous dorsal blastopore lip developed in
the subequatorial dorsal region (Figs. 8A-F; 9A-E). The
onset of neurulation began before completion of blasto-
pore closure (Figs. lOA-D; llA-F).
Developmental time, gastrulation and body elonga-
tion. Our comparative analysis includes frog species
with rapid and slow development (Table 1). Embryonic
development occurs rapidly in frog species with aquatic
reproductive modes. The analyzed frogs with rapid de-
velopment and embryos suspended on the vegetation in-
cluded H. fleischmanni, E. callistomma (Centrolenidae).
Frogs with aquatic eggs and embryos included X. laevis
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Table 2. Characteristics of development of the glass frogs Hyalinobatrachium fleischmanni and Espadarana callistomma.
G
Morphology observed in Centrolenid frogs^
1
Fertilization (not available).
2
Gray crescent (not available).
3
Two cell stage (not available).
4
Four cell stage. The first two cleavage furrows passed from the animal to the vegetal pole. This stage was available only for E. callis-
tomma (not shown).
5
Eight cell stage. The third cleavage furrow was latitudinal in some embryos and longitudinal in others. This stage was available only for
E. callistomma (not shown).
6
Sixteen cell stage. Cleavage became asynchronous after the eight cell stage, and embryos with variable numbers of blastomeres were
observed. This stage was available only iov E. callistomma (not shown).
7
Thirty-two cell stage. Cleavage in both species was holoblastic, and the animal micromeres were much smaller than the vegetal macro-
meres, as observed for other frogs. (Figs. 2A; 4A; 6A-B; 7A-B).
8
Mid cleavage. Development of the blastocoel began during cleavage, as shown for 77. fleischmanni. (Figs. 2B; 6C-D).
9
Blastula. The blastocoel roof was thick and consisted of several cell layers (Figs. 2C; 4B; 7C-D).
10
Early gastrula. A conspicuous blastopore groove was observed on the dorsal subequatorial region of the embryo, and there were bottle
cells marking cell ingression at the blastopore groove as shown for both species (Figs. 4C; 8A; 9A-B). In slightly more advance embryos,
the dorsal blastopore lip was detected in the dorsal subequatorial region, as shown for 77. fleischmanni (Figs. 2D; 8B).
11
Mid gastrula. The blastopore lip surrounded a large yolk plug in embryos of both frogs (Figs. 2E; 4D; 8C). Internally, the archenteron was
elongated, without inflation (Figs. 8D; 9C). The blastocoel roof was translucent (Fig. 1C) and consisted of two-cell layers (not shown).
12
Late gastrula and development of the neural plate (Eigs. 2E; 4E; 8E). The neural groove and the neural plate were visible in gastrula
stage embryos with a small yolk plug (stage 12.5) (Eigs. 2G; 4E, 11 A). The archenteron was elongated in an anterior direction and it
was inflated, and the blastocoel was reduced in size. The cleft of Brachet, that separates the ectoderm from the endomesoderm, was vis-
ible in the roof of the primitive gut (Eigs. 8E; 9D-E; lOA; 11 B-C) The notochord was detected in stage 12.5 embryos, as shown for 77.
fleischmanni (Eig. lOB). In stage 12.75, the neural plate was visible in both species (Eigs. 2H; 4G; 11 D). The yolk plug was small, the
archenteron was fully inflated, and the germ layers were visible (Fig. lOC-D; 1 1 E-F). A triangular dorsal structure, considered to be the
gastrocoel roof plate (grp), was located in the roof of the primitive gut, and was exposed to the cavity of the gastrocoel (Fig. 12C). The
grp included the ventral surface of the notochord and paraxial mesoderm, and was bordered by the lateral endodermal crests (lec). The
grp is illustrated for E. callistomma (Figs. 12D).
13
The closed blastopore and the neural plate. The yolk plug was totally retracted, the blastopore was at the slit blastopore stage, and the
neural plate was visible (Figs. 4H; 12A). The grp was located in the roof of the primitive gut, and it was bordered by the lec, shown in
whole mount for 77. fleischmanni (Fig. 12B).
14
Early neural fold stage. The neural folds were slightly elevated (Eigs. 21; 41; 13A). The grp included the ventral surface of the notochord,
and somites, and it was bordered by the lec, shown forE. callistomma (Eig. 12 E-E). The neural ectoderm, paraxial mesoderm, notochord,
and endoderm were visible (Eig. 13B).
15
Mid neural fold stage. The neural folds were elevated (Eigs. 2J; 4J; 13C). In cross sections, the neural ectoderm, notochord, paraxial
mesoderm and endoderm were visible, as shown for 77. fleischmanni (Eig. 13 D).
16
Closure of the neural tube. The neural folds were closed (Eigs. 2K; 4K; 13 E). In cross sections, the neural tube was visible dorsal to the
notochord. The somites were visible on each side. The endoderm completely lined the archenteron, as shown for E. callistomma (Eigs.
13 E).
17
Tail bud stage. The tail bud and the head region protruded beyond the yolky endoderm. The branchial arches were visible (Eigs. 2L; 4L).
18
Muscular activity. The branchial arches protruded on the sides of the head. The tail became elongated. This stage is only shown for E.
callistomma (Eig. 5A).
19
Heart beat. The heart heated, and the gill buds were visible. This stage is only shown for E. callistomma (Eigs. 5B, C).
20
Circulation to the external gills. There were two gill pairs, each with two small branches. This stage is not shown.
21
The gills were larger, the first pair gill had two branches for both species and the second pair gill was unbranched in 77. fleischmanni
(Eigs. 3A; 5D).
22
Tail fin circulation. Not observed.
23
The external gills reached their full size. There were five gill branches in the first pair and four branches in the second pair of external
gills in embryos of 77. fleischmanni. The opercular fold was developing. There were four gill branches in the first pair and three branches
in the second pair of external gills of E. callistomma embryos (Eigs. 3B-5E).
24
Larval stage. Not observed
25
Tadpole at hatching. Only a small portion of the external gills protruded from the opercular aperture in the hatching tadpoles. The eyes
were very small. (Eigs. 3C-D; 5E).
^ The development of the Centrolenid frogs (C), H. fleischmanni and E. callistomma, was compared with the general staging table
for frogs (G) (Gosner, 1960).
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Hyalinobatrachium fleischmanni
Fig. 3. External morphology of Hyalinobatrachium fleischmanni embryos from the development of the gills stage to hatching. (A)
Stage 21: The gills were large, and each gill pair had two branches. (B) Stage 23: Full development of the external gills. There were
five gill branches in the first pair and four branches in the second pair of gills. (C) Stage 25: Lateral view of a tadpole at hatching.
The eyes were very small. (D) Stage 25: Ventral view of a tadpole at hatching. Only a small portion of the external gills protruded
from the opercular aperture. The pink color of the embryo in A was an artifact of fixation, e, eye; fg, first pair gills; g, gills; mo,
mouth; sg, second pair gills; tf, tail fin.
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Early development of Hyalinobatrachium fleischmanni and Espadarana callistomma
E. callistomma
A B ^ Q 10 D St 11
Fig. 4. External morphology of Espadarana callistomma embryos from cleavage to the tail bud stage. (A) Stage 7: Thirty-two cell
stage. Animal micromeres were much smaller than the vegetal macromeres. (B) Stage 9: Blastula. (C) Stage 10: Early gastrula. A
conspicuous blastopore groove was observed on the dorsal subequatorial region of the embryo. (D) Stage 11: Mid gastrula. The
blastopore lip surrounded a large yolk plug. (E) Stage 12: Late gastrula. (F) Stage 12.5: Late gastrula with a small yolk plug. (G)
Stage 12.75: Late gastrula with a very small yolk plug. The neural plate was visible. (H) Stage 13: The neural plate was visible.
The yolk plug was totally retracted and the blastopore was at the slit blastopore stage. (I) Stage 14: Early neural fold. The neural
folds were visible. (J) Stage 15: Mid neural fold. The neural folds were elevated. (K) Stage 16. Closure of the neural tube. The
neural folds were closed. (L) Stage 17. Tail bud stage. The branchial arches were visible, bg, blastopore groove; hr, branchial arch;
bp, closed blastopore; c, cleavage furrow; dl, dorsal blastopore lip; hy, hyoid arch; ma, mandibular arch; nf, neural fold; ng, neural
groove; np, neural plate; vl, ventral blastopore lip; yp, yolk plug.
(Pipidae), and Ceratophrys stolzmanni (Ceratophryidae),
and frogs with embryos placed in flotating foam-nests
were Engystomops randi and Engystomops coloradorum
(Leptodactylidae) (Table 1). In contrast, embryonic de-
velopment was much slower in embryos of frogs with
terrestrial adaptations. Frogs with slow development
included the Marsupial frog Gastrotheca riobambae
(Hemiphractidae) that broods its embryos in a dorsal
pouch of the mother and the dendrobatid frogs Epipedo-
bates machalilla and Epipedobates tricolor (Dendrobati-
dae) that deposit their eggs in terrestrial nests (Table 1)
(del Pino et al. 2007; Elinson and del Pino 2012).
Gastrulation characteristics vary among frogs accord-
ing to their developmental speed. Gastrulation and body
elongation, as detected by the onset of notochord elonga-
tion, overlapped in embryos of X. laevis, C. stolzmanni.
E. randi, and E. coloradorum, frogs with rapid develop-
ment (Table 1). Similarly, elongation of the notochord
overlapped with gastrulation in the rapidly developing
embryos of the centrolenid frogs H. fleischmanni and E.
callistomma (Figs. 8D, F; 9C-F; lOA-D; IIB-C, E-F;
12D; Table 2). In contrast, gastrulation movements oc-
curred before the onset of notochord elongation in the
slowly developing dendrobatids E. machalilla and E. tri-
color, and in the Marsupial frog, G. riobambae. Egg size
is larger in these slowly developing frogs in comparison
with the rapidly developing species (Table 1), (Elinson
and del Pino 2012; del Pino et al. 2007).
The modular nature of gastrulation allows the separa-
tion of dorsal convergence and extension, the mechanism
that triggers elongation of the notochord and the body,
from gastrulation in the slowly developing frogs, and
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E. callistomma
Fig. 5. External views of Espadamna callistomma embryos from the stage of muscular activity to hatching stages. (A) Stage 18:
Muscular activity. The branchial arches protruded on the sides of the head. (B) Stage 19: Heartbeat. The gill buds were visible. (C)
Stage 19.5: Two gill pairs were visible, each with two small branches. (D) Stage 21: The gills were larger, and each gill pair had two
branches. (E) Stage 23: Full development of the external gills. There were four gill branches in the first pair and three branches in
the second pair of gills. (F) Stage 25: Tadpole at hatching. The eyes were very small, hr, branchial arch; e, eye; fg, first gill pair; gb,
gill bud; hy, hyoid arch; ma, mandibular arch; sg, second gill pair; tf, tail fin.
the overlap of these two processes in rapidly develop-
ing frog species (Elinson and del Pino 2012). Overlap of
gastmlation and body elongation is associated with rapid
development in the unstable conditions of the reproduc-
tive modes that involve aquatic reproduction of X. laevis
and C. stolzmanni, floating foam-nest development in
Engystomops, and suspension of eggs on the vegetation,
in the case of centrolenids frogs (Table 1), (Elinson and
del Pino 2012). The distinct modes of gastmlation likely
relate to the reproductive mode of frogs, rather than to
phylogenetic relationships.
The gastrocoel roof plate (grp) and left-right asym-
metry. It was of interest to determine whether frogs with
different reproductive modes, and different onset of no-
tochord elongation share the pattern of left-right asym-
metry determination by cilia driven fluid flow towards
the left side in the grp, described for X. laevis (Blum et
al. 2014b; Saenz-Ponce et al. 2012b). The question is
particularly important because the mechanism of sym-
metry breakage by cilia driven fluid flow in the grp or
equivalent stmctures is universal among vertebrates with
exception of the chick and the pig (Blum et al. 2014a,b).
In all frogs analyzed, the gastrocoel roof plate (grp) had
a triangular shape and was detected in the dorsal lining
of the primitive gut of the late gastmla and neumla, as
detected in H. fleischmanni and E. callistomma embryos
(Pig. 12A-P; Table 1). As in A laevis and other frogs, the
grp of H. fleischmanni and E. callistomma embryos con-
sisted of the ventral surface of the posterior notochord
and paraxial mesoderm, and it was bordered by the later-
al endodermal crests (lee), illustrated for E. callistomma,
(Pigs. 12D-E). However, in a more rostral region, only
the notochord was exposed to the cavity of the primitive
gut because the paraxial mesoderm was already covered
by the closing lee (Fig. 12F). The major difference de-
tected among frogs was the presence of the grp already
in the late gastmla of the centrolenid frogs, as shown for
E. callistomma (Fig. 12D), whereas the grp developed in
the neumla of X. laevis (Blum et al. 2014b). The preco-
cious onset of grp formation may relate to the overlap of
neumlation and gastmlation in centrolenid frogs, another
example of the modular nature frog gastmlation.
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H. fleischmanni
Fig. 6. Cleavage in Hyalinobatrachium fleischmanni. (A) Stage 7: Animal view of a 32-cell embryo. (B) Stage 7: The blastocoel
of a 32-cell embryo, observed in a sagittal bisection. (C) Stage 8: Animal view of an embryo at mid-cleavage. (D) Stage 8: The
blastocoel of a mid-cleavage embryo, observed in a sagittal bisection, bl, blastocoel.
The grp was detected in the neumla of eight frog spe-
cies with a wide range of reproductive adaptations, and
belonging to six different frog families, (Table 1) (Saenz-
Ponce et al. 2012a, b). The presence of the grp in this
wide range of frogs suggests that determination of left-
right asymmetry may follow mechanisms similar to those
described for X. laevis. Moreover, cilia were detected in
the grp epithelium that lines the dorsal roof of the primi-
tive gut of these various frogs (Saenz-Ponce et al. 2012a,
b). The presence of cilia in the grp in centrolenid frogs
was not analyzed.
Conclusions. The reproductive and developmental strat-
egies of the two centrolenid frogs, analyzed in this work,
differ from each other. The eggs of E. callistomma, de-
posited on the upper sides of plant leaves, contain dark
pigment, and take twice as long to reach the hatching
stage in comparison with H. fleischmanni embryos. In
contrast, the H. fleischmanni development on the under-
side of plant leaves is accompanied by the lack of dark
pigment in the egg and embryos and reduced develop-
mental time. As in other frogs with rapid development,
there was overlap between gastrulation and body elonga-
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E. callistomma
Fig. 7. Cleavage in Espadarana callistomma. (A) Stage 7: Animal view of a 32-cell embryo. (B) Stage 7: The blastocoel of a
32-cell embryo, observed in a sagittal bisection. (C) Stage 9: Animal view of a blastula. (D) Stage 9: The blastocoel of a blastula,
observed in a sagittal section. The blastocoel roof consisted of several cell layers, bl, blastocoel.
tion. Moreover, the process of neurulation already started
during gastrulation, and the grp became visible in the late
gastmla. Presence of the grp in embryos of these cen-
trolenid frogs suggests that the mechanisms of left-right
asymmetry is likely similar with the cilia-driven pattern
of the X. laevis grp.
Acknowledgments. — We thank the Centre of Am-
phibian Investigation and Conservation, Balsa de los Sa-
pos, Pontificia Universidad Catolica del Ecuador (PUCE)
for the donation of embryos of the two species analyzed
in this work. We express gratitude to the members of the
Laboratory of Developmental Biology of PUCE for their
assistance in the conduction of this study, and in particu-
lar we express gratitude to Natalia Saenz-Ponce, Alexan-
dra Vargas, and Andres Garces for their help. We thank
Santiago Ron for providing the photographs of the adults
of both species, and Clifford Keil for critical analysis
of the manuscript and language revision. This study re-
ceived the support of a 2013 research grant from PUCE.
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Hyalinobatrachium fleischmanni
Fig. 8. Gastrulation of Hyalinobatrachium fleischmanni (Stages 10-12). Embryos in A, C, E were stained for cell borders. (A) Stage
10: Early gastrula. Dorsal subequatorial region. The dorsal blastopore groove was visible between the small cells of the animal
region with clearly delineated borders, and the vegetal cells, whose borders were not as clear. (B) Stage 10.5: Sagittal section of an
early gastrula. The dorsal blastopore lip was visible. (C) Stage 1 1 : Mid gastrula. Higher magnification of the dorsal blastopore lip
region. There was difference in size of animal and vegetal cells. (D) Stage 1 1 : Sagittal section of a mid gastrula. The archenteron was
elongated, and the blastocoel roof was reduced to about two cell layers. (E) Stage 12: Late gastrula. Higher magnification of the yolk
plug region. (F) Stage 12: Sagittal section of late gastrula. The arrow indicates the cleft of Brachet. a, archenteron; bg, blastopore
groove; bl, blastocoel; dl, dorsal blastopore lip; vl, ventral blastopore lip; yp, yolk plug.
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E. callistomma
Fig. 9. Gastrulation of Espadarana callistomma (Stages 10-12). (A) Stage 10: Sagittal section of an early gastrula. The dorsal blasto-
pore groove was visible. (B) Stage 10. Higher magnification of the embryo in A, stained for cell nuclei. The arrow indicates a bottle cell
of the blastopore groove area. (C) Stage 11: Sagittal section of mid gastrula. (D) Stage 12: Sagittal bisection of late gastrula. (E) Stage
12: Sagittal section of the late gastrula shown in D. The single cavity is an artifact of sectioning, it corresponds to the blastocoel
and archenteron, as shown in D. (F) Higher magnification of the archenteron roof from the embryo in E, stained for cell nuclei. The
arrow indicates the cleft of Brachet. a, archenteron; bl, blastocoel; dl, dorsal blastopore lip; ec, ectoderm; vl, ventral blastopore lip;
yp, yolk plug.
H. fleischmanni
A st12.5|B St 12.5
Fig. 10. Gastrulation of Hyalinobatrachium fleischmanni (Stages 12.5-12.75). (A) Stage 12.5: Sagittal section of a late gastrula. (B)
Stage 12.5: Cross section through the rostral region of a late gastrula, stained for cell nuclei. The endoderm covered the notochord in
this rostral section. (C) Stage 12.75: Sagittal section of a late gastrula. (D) Stage 12.75: Higher magnification of the archenteron roof
from the embryo in E, stained for cell nuclei. The three germ layers were visible, a, archenteron; dl, dorsal blastopore lip; ec, ectoderm;
en, endoderm; m, mesoderm; pm; paraxial mesoderm, vl, ventral blastopore lip; yp, yolk plug.
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Early development of Hyalinobatrachium fleischmanni and Espadarana callistomma
E. callistomma
Fig. 11. Gastrulation of Espadarana callistomma (Stages 12.5-12.75). (A) Stage 12.5: Late gastrula with a small yolk plug. (B) Stage
12.5: Sagittal section of a late gastrula. (C) Stage 12.5: Sagittal section of the archenteron roof through the rostral region of a late gas-
trula, stained for cell nuclei. (D) Stage 12.75: Late gastrula. The neural plate was visible. (E) Stage 12.75: Parasagittal section of a late
gastrula. (F) Stage 12.75: Higher magnification of the archenteron roof from the embryo in E, stained for cell nuclei. The arrows in C, E
and E indicate the cleft of Brachet. a, archenteron; cbc, circumblastoporal collar; dl, dorsal blastopore lip; ec, ectoderm; en, endoderm;
m, mesoderm; ng, neural groove; np, neural plate; vl, ventral blastopore lip; yp, yolk plug.
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H. fleischmanni
Fig. 12. The gastrocoel roof plate (grp) in embryos of Hyalinobatrachium fleischmanni and Espadarana callistomma. (A) Stage 13:
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H. fleischmanni
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Maria- Jose Salazar-Nicholls is research assistant in the Laboratory of Developmental Biology at the Pon-
tificia Universidad Catblica del Ecuador (PUCE) in Quito. She graduated with a Licenciatura in Biological
Sciences from PUCE in 2013. Her research centers on the characterization of early development in centro-
lenid frogs. She is currently investigating the mode of somitogenesis in Hyalinobatrachium fleischmanni
and Espadarana callistomma. She is interested in climate change and its impacts on conservation.
Eugenia M. del Pino is Professor of Biological Sciences (retired) at PUCE in Quito. She studies the re-
production and development of Marsupial frogs (Hemiphractidae) in comparison with Xenopus laevis, the
model organism of frog developmental biology and with tropical frogs from Ecuador. Her studies are done
in collaboration with PUCE students. The analyses of development reveal important variation in develop-
mental speed according to the reproductive mode of the various frogs. The developmental data is signifi-
cant for the comparative analysis of frog early embryonic development and provide base line information
about the biology of several frog species.
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Official journal website:
amphibian-reptile-conservation.org
Amphibian & Reptiie Conservation
8(1) [Special Section]: 107-120 (e89).
A new species and country record of threadsnakes
(Serpentes: Leptotyphlopidae: Epictinae)
from northern Ecuador
i>2,5David Salazar-Valenzuela, ^Angela Martins, "^Luis Amador-Oyola, and ^Omar Torres-Carvajal
'^Department of Evolution, Ecology and Organismal Biology, Ohio State University, 300 Aronojf Laboratory, 318 W. 12th Avenue, Columbus, Ohio
43210-1293, USA ^Museo de Zoologla, Escuela de Ciencias Biologicas, Pontificia Universidad Catolica del Ecuador, Avenida 12 de Octubre y
Roca, Apartado 17-01-2184, Quito, ECUADOR ^Universidade Eederal do Rio de Janeiro, Museu Nacional, Departamento de Vertebrados, Rio
de Janeiro, Rio de Janeiro 20940-040, BRAZIL “^Departamento de Investigacion Cientlfica, Tecnologica e Innovacion, Universidad Laica Vicente
Rocafuerte, Avenida de Las Americas, Apartado 11-33, Guayaquil, ECUADOR
Abstract . — ^We describe a new species of Triiepida Hedges 2011 from cloud forests of the extreme
northern Ecuadorian Andes, Carchi province. Among other characters, the new species is
distinguished from all congeners by having a subhexagonal ocular with its anterior border barely
rounded at eye level, rostral reaching the anterior border of ocular scales in dorsal view, three
supralabials, four or five infralabials, thicker body width, 203-214 middorsal scales, 12 scales
around middle of tail, uniform gray dorsum, and gray venter with interspaces between scales cream.
Morphologically, the new species is most similar to T. guayaquilensis and T Joshuai from Ecuador
and Colombia, respectively. We also report the first records of T. macroiepis ior the country from the
lowland and foothill evergreen forests of northwestern Ecuador.
Key words. Andes, Choco, cloud forest, fossorial, external morphology, osteology, Triiepida macrolepis; Triiepida
pastusa, new species
Citation: Salazar-Valenzuela D, Martins A, Amador-Oyola L, Torres-Carvajal O. 2015. A new species and country record of threadsnakes (Serpentes:
Leptotyphlopidae; Epictinae) from northern Ecuador. Amphibian & Reptile Conservation 8{t) [Special Section]: 107-120 (e89).
Copyright: © 2015 Salazar-Valenzuela et al. This is an open-access article distributed under the terms of the Creative Commons Attribution-Non-
CommercialNoDerivatives 4.0 International License, which permits unrestricted use for non-commercial and education purposes only, in any medium,
provided the original author and the official and authorized publication sources are recognized and properly credited. The official and authorized
publication credit sources, which will be duly enforced, are as follows: official journal title Amphibian & Reptile Conservation] official journal website
<amphlblan-reptlle-conservatlon.org>.
Received: 12 January 2015; Accepted: 08 February 2015; Published: 02 March 2015
Introduction
Fossorial snakes of the family Leptotyphlopidae are
among the least known terrestrial vertebrates (Adal-
steinsson et al. 2009). Even though some species in the
family may be locally abundant and the group has a wide
distribution from sea level to mountaintops in Africa, the
Americas, and parts of Asia, their secretive habits make
them rarely encountered in the field (Curcio et al. 2002;
McDiarmid et al. 1999; Passes et al. 2005; Pinto et al.
2010). Most leptotyphlopids are small (150 to 250 mm
snout- vent length), thin, and burrowing animals that feed
on social insects (termites are probably the main food
source for some species) (Vitt and Caldwell 2013). Re-
cent phylogenetic analyses based on molecular data parti-
tioned the 1 12 species now recognized in the family in 12
genera (Adalsteinsson et al. 2009; Wallach et al. 2014).
Correspondence. Email: ^davidsalazarv® gmail.com
Amphib. Reptile Conserv.
As a combination of limited morphological variation in
fossorial squamates and paucity of specimens, morpho-
logical synapomorphies for these lineages have not been
clearly established yielding differences in researchers’
opinions as to which genus some species should be allo-
cated. Especially problematic has been the classification
of threadsnakes in the Neotropical genera Rena, Siagon-
odon, and Triiepida (Pinto and Curcio 2011; Pinto and
Eernandes 2012).
In Ecuador, four species of threadsnakes have been
confirmed on the basis of voucher specimens: Epic-
tia signata, E. subcrotilla, Triiepida anthracina, and
T guayaquilensis (Cisneros-Heredia 2008; Pinto et al.
2010; Salazar-Valenzuela et al. 2010; Torres-Carvajal et
al. 2014; Wallach et al. 2014). With the exception of E.
subcrotilla, which seems to be a common taxon in natural
history collections (Cisneros-Heredia 2008; Purtschert
107
March 2015 I Volume 8 | Number 1 | e89
Salazar-Valenzuela et al.
2007), most of the leptotyphlopid species reported for
the country are known from a few specimens or exclu-
sively from the holotype in the case of T. guayaquilensis
(Cisneros-Heredia 2008).
Cryptozoic species of snakes are difficult to find and
the usefulness of regularly including digging techniques
during herpetological surveys has been proposed for fos-
sorial reptiles (Measey 2006). While performing field-
work on the extreme northern Andes of Ecuador, we
found specimens of an undescribed species of Trilepida
with the help of local people who usually dig for archae-
ological remains of Los Pastos pre-hispanic culture. Here
we recognize this species based on morphological data,
increasing the number of species of Trilepida to 14 (Uetz
and Hosek 2014; Wallach et al. 2014). While reviewing
material for this study we came across specimens assign-
able to Trilepida macrolepis, which constitute the first
record of this species for Ecuador and are also reported
herein.
Materials and Methods
We examined specimens housed in the Museo de Zo-
ologia, Pontificia Universidad Catolica del Ecuador
(QCAZ) and the Museo Ecuatoriano de Ciencias Natu-
rales (DHMECN), Quito, Ecuador. In addition, we ana-
lyzed photographs of specimens deposited in the Nation-
al Museum of Natural History, Smithsonian Institution
(USNM), Washington, D.C., USA, and the Museum fur
Naturkunde (ZMB) Berlin, Germany. Characters used
for description and comparisons were based on internal
(skull of a MicroCT Scanned specimen. X-ray plates) and
external morphology (meristic and morphometric data,
shape of cephalic plates, and color pattern) of examined
individuals, as well as published data provided by Ro-
jas-Morales and Gonzalez-Duran (2011) and those sum-
marized in Pinto and Eernandes (2012). We consider the
unique combination of morphological characters present
in the new species as delimitation criteria, following the
general species concept of de Queiroz (1998, 2007). Ter-
minology for cephalic plates, scale features, cloacal sacs,
and measurements follows Broadley and Wallach (2007),
Kroll and Reno (1971), Passos et al. (2006), Pinto and
Curcio (2011), and Pinto and Eernandes (2012). Color
description in life is based on analysis of a series of pho-
tographs of the holotype and paratypes. Color terminolo-
gy and codes follow Kohler (2012). Measurements were
taken with a dial caliper to the nearest 0. 1 mm, except for
total length (TL) and tail length (TL), which were mea-
sured with a ruler to the nearest 1 .0 mm. Measurements
and descriptions of paired cephalic scales are provided
for the right side. Sex was determined by the presence or
absence of hemipenial muscles through a ventral incision
at the base of the tail. Characters recorded are: (1) Total
length (TL); (2) tail length (TAL); (3) TL/TAL ratio; (4)
middorsal scales (rostral and terminal spine excluded);
(5) midventral scales (mental scale, cloacal shield, and
subcaudals excluded); (6) subcaudal scales (terminal
spine excluded); (7) dorsal scale rows around the mid-
dle of the tail (DSR); (8) midbody diameter (MB); (9)
midtail diameter (MT); (10) head length (HE); (11) head
width (HW); (12) relative eye diameter (ocular width at
eye level/eye diameter); (13) presence of fused caudals;
(14) relative rostral width (rostral width/head width)
(Pinto and Curcio 2011; Pinto et al. 2010). The head of
one preserved specimen (QCAZ 5778) was scanned on
a Skyscan 1176 in-vivo high-resolution micro-CT scan
at Universidade de Sao Paulo, Brazil. The specimen was
scanned at 40 kV and 533 uA, and the dataset was ren-
dered in three dimensions through the use of CTVox for
Windows 64 bits version 2.6. Additionally, the skeleton
of the holotype and paratypes were examined dorsally
and ventrally through X-ray plates. Digital radiographies
of QCAZ 5778 were made with a Eaxitron X-Ray EEC
MX 20 at Departamento de Vertebrados, Museu Nacio-
nal, Universidade Rio de Janeiro, Brazil and X-ray scans
of QCAZ 8990 and QCAZ 5846 were made with a Ther-
mo Kevex X-ray Imaging System at QCAZ. Terminol-
ogy for the braincase, mandible, and vertebral column
follows Rieppel et al. (2009), Kley (2006), and Holmann
(2000), respectively.
Results
Trilepida pastusa sp. nov.
urn:lsid:zoobank.org:act:E7C8FFlC-07E8-4985-B673-80A52DACA8Dl
Eigs. 1-3.
Holotype. — Adult female, QCAZ 8690, collected on
23 Eebruary 2009 by O. Torres-Carvajal, S. Aldas-
Alarcon, E. Tapia, A. Pozo and local people, surround-
ings of Chilma Bajo on the way to Tres Marias waterfall
(0°51’53.82” N, 78°2’59.23” W; 2071 m), Tulcan Coun-
ty, Carchi province, Ecuador.
Paratypes. — Two specimens with same locality data as
holotype: one juvenile female (QCAZ 5778) collected on
21 Eebruary 2013 by D. Salazar-Valenzuela, H. Pozo, A.
Chalapud, and D. Males, and one juvenile of undeter-
mined sex (QCAZ 5846) collected on 20 March 2013 by
D. Salazar-Valenzuela and A. Loaiza-Lange.
Diagnosis. — Trilepida pastusa is distinguished from all
congeners by the following combination of characters:
Snout truncate in dorsal and ventral view, rounded in
lateral view; supraocular present; ocular subhexagonal
with superior border straight and anterior border barely
rounded at eye level; rostral subtriangular in dorsal view,
reaching anterior border of ocular scales; frontal as long
as supraocular and other middorsal cephalic shields, ex-
cept for postfrontal which is smaller; temporal distinct;
supralabials three (2+1); infralabials four or five; body
March 2015 I Volume 8 | Number 1 | e89
Amphib. Reptile Conserv.
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A new species of threadsnake from northern Ecuador
Fig. 1. Dorsal (A), lateral (B), and ventral (C) views of the head
of the holotype of Trilepida pastusa sp. nov. (QCAZ 8690).
Scale bar =1.5 mm. Drawings by D. Paucar.
width relatively thick (TL/MB = 28.6-34.6); middorsal
scales 203-214; midventral scales 182-193; subcaudal
scales 18-19; fused caudals present; terminal spine coni-
cal, slightly longer than wide; scales around middle of
tail 12; dorsum uniform gray; venter gray with interspac-
es between scales cream.
Comparisons (Table 1). — Among all congeners, Trilep-
ida pastusa is more similar to T guayaquilensis and T.
joshuai in sharing 12 scales around midtail, three supra-
labials, and a uniform dark dorsum and pale venter (Pin-
to and Fernandes 2012). The new species differs from
both by having an ocular with an anterior border barely
rounded at eye level, a thick body (TL/MB = 28.6-34.6),
and a coloration pattern consisting of a uniform gray dor-
sum and a venter with gray on the center and cream on
the outside of each scale (Figs. 3, 4A) (vs ocular with
rounded anterior border, thin body [TL/MB = 48.6], and
uniform brown color dorsally and pale brown ventrally in
T. guayaquilensis; ocular with rounded anterior border,
moderate to thin body width [TL/MB = 34.0-55.2], and
uniform dark brown dorsally and cream ventral scales
in T. joshuai) (Orejas-Miranda and Peters 1970; Pinto
and Fernandes 2012; Pinto et al. 2010; Rojas-Morales
and Gonzalez-Duran 2011). Moreover, T. pastusa dif-
fers from T. guayaquilensis in having a lower number of
middorsal (203-214 vs 253, respectively) and midventral
(182-193 vs 233, respectively) scales (Orejas-Miranda
and Peters 1970; Pinto and Fernandes 2012). The new
species also differs from T. joshuai by having a higher
number of middorsal (203-214 vs 174-199, respective-
ly) and midventral (182-193 vs 165-187, respectively)
scales, and a higher number of subcaudals (18-19 vs 13-
18, respectively) (Pinto and Fernandes 2012; Pinto et al.
2010; Rojas-Morales and Gonzalez-Duran 2011). Some
specimens of T. macrolepis can have 12 scales around
Table 1. — Meristic and morphometric variation of the four species of Trilepida occurring in Ecuador. Data are from Pinto and Fer-
nandes (2012) and references therein, as well as our own scale counts and measurements. Abbreviations: DO = middorsal scales; VE
= midventral scales; SC = subcaudals; TL = total length; TAL = tail length; MB = midbody diameter; MT = midtail diameter; SL =
supralabials; IL = infralabials; SO = supraocular. Color pattern adapted from Passos et al. (2006) and Pinto and Fernandes (2012):
1 = uniform violet black dorsally and ventrally; 2 = reticulate dark brown dorsally and reticulate pale brown ventrally; 3 = uniform
brown dorsally and pale brown ventrally; 4 = uniform gray dorsally and reticulate gray ventrally.
Character
Trilepida pastusa sp. nov.
Trilepida macrolepis
Trilepida guayaquilensis
Trilepida anthracina
DO
203-214
211-255
253
182-193
VE
182-193
201-237
233
167-176
SC
18-19
16-24
20
15-19
TL/TAL
10.86-13.67
8.2-15.9
13.1
12.2-16.6
TL/MB
28.63-34.55
32.2-68.3
48.6
31.7-43.7
TAL/MT
2.67-3.81
3.8-7.9
-
3.6-10.1
SL
2+1
2+1
2+1
2+1
IL
4-5
4
4
4
SO
present
present
present
present
Midtail scales
12
10
12
10
Color pattern
4
2
3
1
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Salazar-Valenzuela et al.
Fig. 2. Dorsal (A) and ventral (B) views of the holotype of Tri-
lepida pastusa sp. nov. (QCAZ 8690) in preservative. Scale bar
= 1 cm.
midtail (see Discussion), but T. pastusa differs from this
species by having an ocular with barely rounded anterior
border, thick body (TL/MB = 28.6-34.6), uniform gray
dorsum and venter with gray on the center and cream
on the outside of each scale, 203-214 middorsal scales,
182-193 midventral scales, 18-19 subcaudals, non-en-
larged eyes, and rostral reaching ocular level in dorsal
view (vs ocular with rounded anterior border, moderate
to thin body width [TL/MB = 32.2-68.3], reticulate dark
brown dorsally and reticulate pale brown ventrally, 211-
255 middorsal scales, 201-237 midventral scales, 16-24
subcaudals, enlarged eyes, and rostral not reaching ocu-
lar level in T. macrolepis) (Fig. 4) (Pinto and Fernandes
2012; Pinto et al. 2010).
Description of the holotype (Figs. 1, 2 ). — Adult female,
TL 315 mm, TAL 29 mm; MB 10.2 mm; MT 7.6 mm;
TL/TAL 10.9; TL/MB 30.9; TAL/MT 3.8; HL 6.2 mm,
HW 6.0 nun; relative eye diameter 3.1; relative rostral
width 0.4. Body subcylindrical, robust, head slightly
compressed compared to body and slightly tapered cau-
dally near tail. Head subcylindrical, as long as wide and
slightly distinguishable from neck. Snout slightly round-
ed in dorsal and ventral views, rounded in lateral view;
rostral straight in frontal and ventral views, subtriangular
in dorsal view but with rounded apex, reaching imagi-
nary transverse line between anterior border of oculars;
rostral contacting supranasal and infranasal laterally, and
Amphib. Reptile Conserv.
frontal dorsally; nasal completely divided horizontally
by oblique suture crossing nostril and descending pos-
teriorly to contact first supralabial; nostril roughly ellip-
tical, obliquely oriented and located in middle of nasal
suture; supranasal higher than wider, bordering rostral
anteriorly, infranasal inferiorly, first and second supral-
abials, and ocular posteriorly, and frontal and supraocu-
lar dorsally; supranasal ventral margin half the length of
upper border of infranasal scale; infranasal about twice
as high as wide, longer than any of the supralabials; up-
per lip border formed by rostral, infranasal, two anterior
supralabials, ocular, and posterior supralabial; temporal
distinct in size from dorsal scales of lateral rows; three
supralabials, first two anterior to ocular and one posterior
(2+1); first supralabial almost twice as high as wide, not
reaching nostril and eye levels, second supralabial almost
twice as high as wide, higher than first supralabial, reach-
ing nostril level; third supralabial trapezoidal, as high as
wide, reaching nostril level, its posterior margin in broad
contact with temporal; ocular enlarged, subhexagonal,
anterior border barely rounded at eye level, higher than
wide, contacting posterior margins of supranasal and sec-
ond supralabial anteriorly, parietal and third supralabial
posteriorly, and supraocular dorsally; eye distinct (diam-
eter = 0.7 nun), located in central area of upper part of
ocular, displaced above nostril level; supraocular longer
than wide, as long as frontal, between ocular and fron-
tal, contacting supranasal anteriorly, frontal and ocular
laterally, and parietal and postfrontal posteriorly; frontal,
interparietal, and interoccipital subequal in size, hex-
agonal and imbricate, postfrontal smaller; frontal longer
than wide, contacting rostral, supranasal, supraocular,
and postfrontal; postfrontal as long as wide, contacting
frontal, supraocular, parietals, and interparietal; interpa-
rietal as long as wide, contacting postfrontal, parietals,
occipitals, and interoccipital; interoccipital wider than
long, contacting interparietal, occipitals, and first dorsal
scale of vertebral row; parietal and occipital subequal, ir-
regularly heptagonal; parietal longer than occipital, twice
as high as wide, lower margin contacting upper border
of third supralabial, posterior margin contacting tempo-
ral, occipital, and interparietal, anterior border in contact
with ocular, supraocular, and postfrontal; occipital twice
as high as wide, its lower limit attaining upper margin of
third supralabial, separated from the latter by temporal;
symphysial trapezoidal, anterior border slightly concave
and posterior border convex except in the middle, five
times wider than high; four infralabials; first infralabial
twice as high as wide; second infralabial as high as wide;
third infralabial twice as wide as high and not pigment-
ed; fourth infralabial as high as wide. Cephalic shields
with uniformly scattered sensory pits. Middorsal scales
203; midventral scales 182; scales rows around middle
of body 14, reducing to 12 rows in middle of tail; cloacal
shield triangular, as wide as long; subcaudals 19; fused
caudals present; terminal spine conical, slightly longer
than wide; elongated cloacal sacs present. Dorsal scales
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March 2015 I Volume 8 | Number 1 | e89
A new species of threadsnake from northern Ecuador
Fig. 3. Trilepida pastusa in life. Lateral view of body (A) and head (B) of holotype (QCAZ 8690) and lateral (C) and ventral (D)
views of body of juvenile paratype (QCAZ 5846). Photographs by O. Torres -Carvajal and S.R. Ron.
homogeneous, cycloid, smooth, imbricate, and wider
than long.
Coloration in preservative of the holotype (Fig. 2 ). —
Middorsal scales (i.e., seven longitudinal rows) bluish
gray. The remaining seven scale rows forming the ventral
and lateral sides of the body are occupied on the center
by the same bluish gray color, but the margins of each
scale are cream white; the latter pattern is less apparent
on the anterior fourth of the body. Border of mouth, men-
tal scale, nostrils and eyes are cream. Cloacal shield blu-
ish gray except on its posterior margin, which is cream
with bluish-gray dots.
Color variation. — Dorsal ground color of body similar to
that of the holotype in one of the juveniles (QCAZ 5846),
the other juvenile (QCAZ 5778) is dark gray; ventral col-
oration is similar in all specimens.
and they turned Pale Neutral Gray (Color 296) (Fig. 3D).
Tongue Smoky White (Color 261).
Quantitative variation. — Scale counts in Trilepida pas-
tusa vary as follows: middorsal scales 203-214 (x =
206.67 + 6.35, n = 3); midventral scales 182-193 (x =
186 + 6.08, n = 3); subcaudals 18-19 (x = 18.67 + 0.58,
n = 3); TL 315 mm (n = 1) in adult and 123-136 nun
(x = 129.5 mm + 9.19, n = 2) 'm juveniles; TAL 29 mm
(n = 1) in adult and 9-12 mm (x = 10.5 mm + 2.12, n =
2) in juveniles; TL/MB ratio 30.94 (n = 1) in adult and
28.63-34.55 (x = 31.59 + 4.19, n = 2) in juveniles; TAL/
MT ratio 3.81 (n = 1) in adult and 2.67-3.41 (x = 3.04 +
0.52, n = 2) in juveniles; infralabials 4 (n = 1) in adult and
5 (n = 2) in juveniles; relative eye diameter 3.07 (n = 1) in
adult and 1.70-1.73 (x = 1.71 + 0.02, n = 2) in juveniles;
relative rostral width 0.37 (n = 1) in adult and 0.31-0.38
(x = 0.35 + 0.05, n = 2) in juveniles.
Color in life. — Dorsum uniform Dark Blue Gray (Color
194) (Figs. 3 A, 3C, 4A), with upper part of head Brick
Red (Color 36) on both juveniles (Figs. 3C, 4A). Venter
of body and tail Dark Blue Gray, with interspaces be-
tween scales Cream White (Color 52) (Figs. 3B, 3D, 4A).
Anal plate entirely Dark Blue Gray. After a few minutes
of handling, the borders of each scale became apparent
Skull (Fig. 5). — Premaxilla roughly rectangular in frontal
and ventral views, edentulous, pierced by six foramina;
transverse process of premaxilla absent and vomerian
process double; nasals paired, approximately quad-
rangular dorsally, and pierced by a pair of foramina in
lateral border of contact with prefrontals; nasal septum
descending as medial vertical flanges; prefrontals paired,
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Salazar-Valenzuela et al.
Fig. 4. Lateral (A) view of body of juvenile paratype of Trilepida pastusa (QCAZ 5778). Lateral (B), dorsal (C), and ventral (D)
views of body of T. macrolepis (DHMECN 11400). Dorsal view of head of the holotype of T. pastusa (QCAZ 8690) (E) and T.
macrolepis (DHMECN 11400) (E). Photographs by L.A. Coloma, O. Torres-Carvajal, and S.R. Ron.
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A new species of threadsnake from northern Ecuador
subtriangular in dorsal view; septomaxillae paired, com-
plex in shape, expanding dorsally into the naris; conchal
invagination absent; ascending process of premaxilla
pierced by single large foramen; vomers paired, located
midventral to vomeronasal cupola, bearing transver-
sal arms, and with short posterior arms; frontals paired,
nearly rectangular dorsally, without anterolateral projec-
tions to attach to prefrontals; frontal pillars absent; optic
nerve restricted to lateral descending surface of frontals;
maxilla edentulous, irregular in shape, pierced by three
large juxtaposed foramina; posterior orbital element ab-
sent; parietal single, wide, representing largest bone of
braincase; parabasisphenoid arrow-like, with tapered
anterior tip lying bellow palatine, and fitting in medial
line of vomeronasal cupola; parabasisphenoid with shal-
low pituitary fossa; basioccipital single and pentagonal;
supraoccipitals fused into single unit, subpentagonal;
prootics paired and triangular; large statolythic mass
present in cavum vestibuli; crista tubelaris not enclos-
ing juxtastapedial recess; stapedial footplate apparently
not co-ossified with prootic; otoccipitals paired and rect-
angular; palatines paired and triradiate; anterior margin
of maxillar process slightly flexing ventrally; pterygoids
slender and rod-like, not contacting quadrate posteriorly,
and not extending beyond the anterior margin of basioc-
cipital; ectopterygoid indistinct; quadrate long and slen-
der, about 50% of skull length; dentary supports a series
of five teeth ankylosed to the inner surface of the antero-
lateral margin of dental concha; mental foramen nearly
under the teeth; splenial conical, representing
smallest bone in lower jaw; anterior mylohyoid foramen
absent on splenial; posterior milohyoid foramen on the
ventral surface of angular; angular conical; compound
bone pierced by two foramina in the surangular lamina,
posterior surangular foramen large and anterior to the
articular portion of compound bone, anterior surangular
foramen located below the coronoid; coronoid rests on
the compound bone.
Postcranial data. — Precloacal vertebrae 176-184 (x =
179 + 4.16, n = 3); cervical vertebrae 6 -i- trunk vertebrae
170 {n = 1); caudal vertebrae 23-24 (x = 23.3 + 0.6, n
= 3), the last vertebrae representing the fusion of three
vertebrae {n = 2). Correlation (n = 3) between middor-
sal scales and precloacal -i- subcaudal vertebrae (1:0.99),
between midventral scales and precloacal vertebrae
(1:1.02), and between subcaudal scales and caudal verte-
brae (0.8:1). Pelvic girdle located at the level of the 17P'
and 176* precloacal vertebrae (QCAZ 5778), or 176*
precloacal and 2"‘^ subcaudal (QCAZ 8690). Pelvic girdle
represented by four non-fused bones: ilium, ischium, fe-
mur, and pubis. Ilium, ischium, and femur rod-like; ilium
represents the longest bone of pelvic girdle; femur stout.
Etymology. — The specific epithet is used as a noun in
apposition. As explained in Coloma et al. (2010), pas-
tuso or pastusa is a Spanish word used to refer to the
inhabitants of the Pasto region in northern Ecuador and
southern Colombia. Here, we also use it to recognize the
presence of Los Pastos pre-hispanic culture (500-1500
CE) (Delgado-Troya 2004), whose vestiges remain in the
type locality and allowed the discovery of Trilepida pas-
tusa and specimens of another cryptozoic snake species:
Atractus savagei (Salazar- Valenzuela et al. 2014).
Proposed standard English and Spanish names. — Pas-
tuso threadsnakes; Serpientes hebra pastusas.
Distribution and natural history (Pigs. 6, 7). — Western
versant of the Cordillera Occidental of extreme northern
Ecuadorian Andes in Tulcan County, Carchi province.
Trilepida pastusa is known only from the type local-
ity, which belongs to Montane Cloud Porest (Valencia
et al. 1999) at 2,071 m. The holotype was found below
mounds of dirt, stones, pasture and moss in conjunction
with eggs of Liophis vitti (nomenclature following rec-
onnnendations expressed in Curcio et al. 2009) and adult
specimens of Atractus savagei (Salazar- Valenzuela et al.
2014). Juvenile paratypes were found below rocks (ca.
40 cm in diameter) in areas of pasture.
First records of Trilepida macrolepis for Ecuador. — The
big-scaled threadsnake, T macrolepis, is a relatively
large (126-322 mm) leptotyphlopid snake with the wid-
est geographical distribution of all species in the genus
(Pinto et al. 2010). Localities for the species in north-
ern South America include Panama, Colombia, Ven-
ezuela, Guyana, Suriname, Prench Guiana, Brazil, and
Peru (Wallach et al. 2014). It is distinguished from con-
geners by having three supralabial and four infralabial
scales, 10 rows in the middle of the tail, more than 210
middorsal scales, and more conspicuously because of a
dorsal and ventral coloration pattern consisting of dark
brown to black scales with white borders (i.e., reticulate)
(Passos et al. 2005; Pinto and Pemandes 2012; Pinto et
al. 2010). Two specimens from Esmeraldas province
in northern Ecuador agree with most of these charac-
ters, their scale counts vary as follows (QCAZ 10247,
juvenile of undetermined sex and DHMECN 11400,
adult male, respectively): middorsal scales 239, 250;
midventral scales 220, 228; subcaudals 21, 20; TL 158
mm, 333 mm; TAP 11 mm, 23 mm; SL 2-1-1, 2-1-1; IP 4,
6; SO 1, 1; midtail scales 12, 10. Coloration pattern on
both is reticulate dark brown dorsally and reticulate pale
brown ventrally (Pigs. 4B^D). Specimen QCAZ 10247
was collected on 13 Pebruary 2010 in Otokiki Reserve,
Alto Tambo (0°54’21.6” N, 78°36’21.6” W, 620 m), San
Lorenzo County, Esmeraldas province; the snake was
found in primary forest 30 cm below ground among fern
roots. Specimen DHMECN 11400 was collected on 01
April 2012 near Durango (1°02’30.7” N, 78°37’26.6”
W, 243 m), San Lorenzo County, Esmeraldas province;
the snake was found in secondary forest one m above
ground among leaf litter accumulated on the junction of
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Salazar-Valenzuela et al.
Fig. 5. Three-dimensional reconstruction of the skull of Trilepida pastusa based on HRXCT data. Dorsal (A), ventral (B), lateral
(C), anterior (D), and posterior (E) views of juvenile paratype (QCAZ 5778). Scale bar = 3.5 mm. Bo, basioccipital; CB, compound
bone; De, dentary; Fr, frontal; Ma, maxilla; Na, nasal; Ot, otico-occipital; Pa, parietal; Pal, palatine; Pbs, parabasisphenoid; Pf,
prefrontal; Pm, premaxilla; Pr, prootic; Pt, pterygoid; Qd, quadrate; Sm, septomaxilla; So, supraoccipital; Vo, vomer.
Amphib. Reptile Conserv.
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A new species of threadsnake from northern Ecuador
lianas. Both localities belong to Lowland and Foothill
Evergreen Forests of northwestern Ecuador (Ceron et al.
1999) (Fig. 6).
Discussion
The conservation assessment of reptile species belong-
ing to families that are completely or primarily fossorial
(e.g., Amphisbaenidae, Anomalepididae, Leptotyphlo-
pidae, Typhlopidae, Uropeltidae) is incomplete (Santos
2013). Due to their secretive habits and non-inclusion
during routine herpetological surveys, knowledge about
their distribution and population dynamics is scarce
(Measey 2006; Pyron and Wallach 2014). Bohm et al.
(2013) estimated that 10.5% (range: 5.6-57%) of species
of fossorial reptiles are Threatened; however, the authors
recognized that this low estimate and wide confidence
intervals reflect the fact that a large number (47% from a
subsample of 1,500 reptile species) of the included taxa
had been classified as Data Deficient. Therefore, this
study re-emphasized the need to target these groups in
future research and surveys.
Records for Trilepida macrolepis and T. pastusa pro-
vided here come from a region where several new spe-
cies of snakes have been discovered in the last 15 years
(e.g., Dixon 2000; Passos et al. 2009; Salazar- Valenzuela
120
ECUADOR
Pacmc
Ocean
PERU
ALTITUDE (m)
I I fl.HM
m Ml - t.OQO
1 . 1 H 1 - 1 ^
V»1 -3.000
2.001 - 2 ^500
^.SOl - 3 j000
>^.000
rii' w
Fig. 6. Geographic distribution of Trilepida pastusa (circle) and
T. macrolepis (triangles) in Ecuador.
Fig. 7. Habitat of Trilepida pastusa surrounding the cloud forests near the town of Chilma Bajo, Tulcan County, Carchi province
(A); pasture areas where individuals were collected (B); microhabitat of T pastusa (C); and a juvenile individual of the new species
in situ (arrow) (D). Photographs by D. Salazar-Valenzuela.
Amphib. Reptile Conserv.
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Salazar-Valenzuela et al.
et al. 2014; Torres-Carvajal et al. 2012). The singularity
of this region may be attributable to the proposed exis-
tence of a habitat transition between northern and cen-
tral parts of the Choco bioregion (western Colombia and
northwestern Ecuador) and communities found further
south along the Pacific coast and adjacent Andean slopes
of Ecuador (Anderson and Jarrin-V 2002; Anderson and
Martmez-Meyer 2004; Cisneros-Heredia 2006; Salazar-
Valenzuela et al. 2014).
Trilepida macrolepis has been suggested to represent
a complex of species due to its wide distribution, pres-
ence on both sides of the Andes, and deep molecular
divergence between individuals from two localities in
northern Brazil and Guyana (Adalsteinsson et al. 2009;
Orejas-Miranda 1967). Our data from Ecuador agree
with the diagnosis provided for this species in Pinto et
al. (2010), except for the presence of 12 midtail scales in
specimen QCAZ 10247. This character has been used in
the taxonomy of members of the genus (Orejas-Miranda
and Peters 1970; Pinto et al. 2010) and will need to be
evaluated in future studies. We tentatively assign QCAZ
10247 to T. macrolepis based mainly on the presence of
a high number of middorsal and midventral scales (239
and 220, respectively), an ocular with rounded anterior
border, and a reticulate dorsal and ventral coloration
pattern, but acknowledge that a detailed revision of this
taxon is needed to confirm the taxonomic identity of this
specimen. Perez-Santos and Moreno (1991) showed two
color photographs of leptotyphlopid snakes of Ecuador
without species identification. One of them (picture 148)
seems to agree with the dorsal reticulate pattern present in
T. macrolepis, suggesting that this taxon was already col-
lected in Ecaudor. However, the same image (picture 85)
was also included in Perez-Santos and Moreno (1988)’s
book on snakes of Colombia rendering questionable the
origin of that specimen. The discovery of individual DH-
MECN 11400 among leaf litter one m above the forest
floor is in agreement with the report of individuals of this
species complex from the Amazonian lowlands wrapped
two m above ground around small tree trunks and mov-
ing their heads back and forth (Vitt and Caldwell 2013).
These authors suggested that the snakes were probably
trying to detect airborne chemical cues associated with
termite nests.
Putative synapomorphies for the genus Trilepida in-
clude a hemipenis body with a narrow base and a robust
terminal portion, middorsal cephalic scales of moderate
size (i.e., supraocular scales smaller or equal to frontal
and postfrontal scales), and an enlarged terminal spine
(Passos et al. 2006; Pinto and Curcio 2011; Pinto and
Fernandes 2012). Although we could not examine hemi-
penis for T. pastusa since adult males were not avail-
able in our sample, we assign this species to the genus
Trilepida based on the presence of middorsal cephalic
scales of moderate size and a slightly enlarged terminal
spine (see Pinto and Curcio, 2011). Also, the paired or
unpaired condition of the nasal bone is variable in differ-
Amphib. Reptile Conserv.
ent members along the Leptotyphlopidae family (Rieppel
et al. 2009). Although osteological characters have not
been employed in less inclusive phylogenetic analysis
on the Renina subtribe (represented by the genus Rena
and Trilepida), previous morphological studies on the
skull of members pertaining to this subtribe (e.g.. Brock
1932; List 1966; McDowell and Bogert 1954; Rieppel et
al. 2009) indicate that the paired condition of the nasal
bones may be a feature that could distinguish the genus
Trilepida (paired condition) from the genus Rena (fused
condition). As many other species currently allocated
in the genus Trilepida, T pastusa also has paired nasal
bones. Intrageneric phylogenetic relationships have not
been established for members of the genus Trilepida', out
of the 14 species of the genus, three consistently show
the presence of 12 midtail scales: T guayaquilensis, T
joshuai, and T pastusa. These species are restricted to
Colombia and Ecuador and these shared features may
indicate close phylogenetic relationships between them.
Trilepida guayaquilensis is still only known from the ho-
lotype even though it was described 45 years ago from a
specimen collected in Guayaquil, Guayas province, Ec-
uador. We analyzed photographs from specimens collect-
ed in this locality and misidentified as T guayaquilensis.
These specimens actually represent Epictia subcrotilla,
which is a leptotyphlopid snake distributed in the low-
lands of Ecuador and Peru, relatively common in natural
history collections (Cisneros-Heredia 2008; Purtschert
2007). Indeed, specimen QCAZ 12769 collected by us
in Guayaquil confirms the presence of this species in ur-
ban areas of this city. Trilepida guayaquilensis therefore
remains to be confirmed as a species with a distribution
that includes Guayaquil but no further records exist from
this area because it is either extremely rare in abundance
or searching efforts have not been enough to locate this
fossorial animal. Alternatively, T. guayaquilensis could
be a species whose only known specimen did not origi-
nate from Guayaquil but may have been brought there
from a nearby locality in the Pacific lowlands or the
western versant of the Andes, as has been suggested for
a couple of other species of Ecuadorian snakes (Cadle
2005; Curcio et al. 2012).
As is common in tropical parts of the world where
these groups have significantly diversified (Pyron and
Burbrink 2012; Vitt and Caldwell 2013), knowledge
about the diversity of fossorial snake fauna in Ecuador is
still fragmented. Cisneros-Heredia (2008) clarified much
of the confusion present at the time regarding the exis-
tence of voucher specimens for species registered in the
country and concluded that only three species of the fam-
ily Leptotyphlopidae were present in the country. The ad-
dition of two species of threadsnakes to the snake fauna
of Ecuador reported in this study should bring us closer
to the true diversity present in the country, even though
it is clear that there are more species that remain to be
described in this group (Cisneros-Heredia 2008, pers.
observ.).
116
March 2015 I Volume 8 | Number 1 | e89
A new species of threadsnake from northern Ecuador
Acknowledgments. — We thank the following cu-
rators and their staff for allowing us to examine speci-
mens or photographs of specimens under their care: K.
de Queiroz and J. Poindexter (USNM) and M. Yanez-
Munoz (DHMECN). We are grateful to E. Levy, A. Pozo,
and the Chilma Bajo connnunity for assistance with lo-
gistics in the field. S. Aldas- Alarcon, G. Buitron, A. Cha-
lapud, A. Loaiza-Lange, D. Males, P. Piedrahita, H. Pozo,
P. Santacruz-Ortega, and E. Tapia provided assistance in
the field. We thank L.A. Coloma for the use of his photo-
graphs of Trilepida macrolepis, R. Pinto for kindly shar-
ing photographs of the holotype of T. guayaquilensis, D.
Paucar-Guerrero and S.R. Ron for illustrations and pho-
tographs of T. pastusa, S. Lobos for improving the dis-
tribution map, and A. Varela for help with figure edition.
Reviews by P. Passes and an anonymous reviewer sub-
stantially improved the manuscript. Specimens were col-
lected under collection permit 008-09 IC-EAU-DNB/MA
and were deposited at Museo de Zoologfa (QCAZ), Pon-
tificia Universidad Catolica del Ecuador. OTC received
support from Secretarfa de Educacion Superior, Ciencia
y Tecnologfa del Ecuador (SENESCYT), project PIC-08-
0000470. Einancial support for AM was provided by the
Coordena^ao de Aperfeigoamento de Pessoal de Nfvel
Superior (CAPES), Conselho Nacional de Desenvolvim-
ento Cientifico e Tecnologico (CNPq), and the Fundagao
de Amparo a Pesquisa do Rio de Janeiro (EAPERJ).
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APPENDIX
Specimens examined
Epictia subcrotilla (n = 4). — ECUADOR: Provincia Guayas: Guayaquil, 7 m, QCAZ 12769; USNM 232401-03
(photographs examined).
Trilepida anthracina (n = 1). — ECUADOR: Provincia Zamora Chinchipe: 6.5 km SE of Tundayme, 1,300-1,500
m, QCAZ 7396.
Trilepida guayaquilensis {n = 1). — ECUADOR: Provincia Guayas: Guayaquil, ZMB 4508 (holotype) (photo-
graphs examined).
Trilepida macrolepis (n = 2). — ECUADOR: Provincia Esmeraldas: Alto Tambo, Otokiki Reserve, 620 m, QCAZ
10247; Durango, 243 m, DHMECN 11400.
David Salazar- Valenzuela graduated in Biological Sciences from Pontificia Universidad Catdlica del Ec-
uador (PUCE) in 2007. He is currently a researcher of the Museo de Zoologia QCAZ of PUCE and a Ph.D.
candidate in the Department of Evolution, Ecology and Organismal Biology at The Ohio State University.
His doctoral dissertation is focused on systematics, historical demography, and venom variation of the
Bothrops asper species complex using genomic and proteomic approaches. So far David has published six
scientific papers on taxonomy, ecology, and toxinology of Ecuadorian amphibians and reptiles.
Angele Martins received ber Master’s degree in 2012 from Museu Nacional do Rio de Janeiro/UFRJ-
Brazil, and is now a Pb.D. student in Zoology at this same institution. She has dedicated her research
efforts in the last six years to the study of snake morphology and herpetofauna from the Atlantic Forest in
Brazil. In the last three years, she has focused on the study of the comparative anatomy of scolecophidians,
with significant interest on threadsnakes (Eeptotyphlopidae), which lead her to investigate the comparative
anatomy of this group for her Ph.D. thesis.
Luis Amador-Oyola graduated in Biological Sciences from the University of Guayaquil (UG) in 2005.
He is currently completing his Master’s thesis on the biogeography and systematics of amphibians from
the Chonghn Colonche mountains (equatorial pacific) at the same university. His work has focused on the
distribution and biodiversity of amphibians and reptiles of areas on the coast of Ecuador, however Luis is
interested in future work on evolution and biogeography of amphibians. This manuscript represents the
first description of a species of herpetofauna coauthored by Luis; other works are in preparation.
Omar Torres-Carvajal graduated in Biological Sciences from Pontificia Universidad Cathlica del Ecua-
dor (PUCE) in 1998, and in 2001 received a Master’s degree in Ecology and Evolutionary Biology from
the University of Kansas under the supervision of Dr. Linda Trueb. In 2005 he received a Ph.D. degree
from the same institution with the thesis entitled “Phylogenetic systematics of South American lizards
of the genus Stenocercus (Squamata: Iguania).” Between 2006-2008 he was a postdoctoral fellow at the
Smithsonian Institution, National Museum of Natural History, Washington DC, USA, working under the
supervision of Dr. Kevin de Queiroz. He is currently Curator of Reptiles at the Zoology Museum QCAZ of
PUCE and an Associate Professor at the Department of Biology in the same institution. He has published
more than 30 scientific papers on taxonomy, systematics, and biogeography of South American reptiles,
with emphasis on lizards. He is mainly interested in the theory and practice of phylogenetic systematics,
particularly as they relate to the evolutionary biology of lizards.
Amphib. Reptile Conserv.
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Salazar-Valenzuela et al.
In accordance with the International Code of Zoological Nomenclature new rules and regulations (ICZN 2012), we have deposited this paper in publicly accessible institutional libraries.
The new species described herein has been registered in ZooBank (Polaszek 2005a, b), the official online registration system for the ICZN. The ZooBank publication LSID (Life Science
Identifier) for the new species described here can be viewed through any standard web browser by appending the LSID to the prefix “http://zoobank.org/”. The LSID for this publication
is: urn:lsid:zoobank.org:pub:3FC7DC45-E3D4-49B6-AEDD-3925A347665E.
Separate print-only edition of paper(s) (reprint) are available upon request as a print-on-demand service. Please inquire by sending a request to: Amphibian & Reptile Conservation
(amphibian-reptile-conservation.org; arc.publisher@gmail.com).
Amphibian & Reptile Conservation is a Content Partner with the Encyclopedia of Life (EOL); http:///www.eol.org/ and submits information about new species to the EOL freely.
Digital archiving of this paper are found at the following institutions: ZenScientist (http://www.zenscientist.com/index.php/filedrawer); Ernst Mayr Library, Museum of Comparative Zool-
ogy, Harvard University, Cambridge, Massachusetts (USA); Florida Museum of Natural History, Gainesville, Florida (USA).
Complete journal archiving is found at: ZenScientist (http://www.zenscientist.com/index.php/filedrawer); Florida Museum of Natural History, Gainesville, Florida (USA).
Citations
ICZN. 2012. Amendment of Articles 8,9,10,21 and 78 of the International Code of Zoological Nomenclature to expand and refine methods of publication. Zootaxa 3450: 1-7.
Polaszek A et al. 2005a. Commentary: A universal register for animal names. Nature 437: 477.
Polaszek A et al. 2005b. ZooBank: The open-access register for zoological taxonomy: Technical Discussion Paper. Bulletin of Zoological Nomenclature 62(4): 210-220.
Amphib. Reptile Conserv.
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Number 1
e89
Official journal website:
amphibian-reptile-conservation.org
Amphibian & Reptiie Conservation
8(1) [Special Section]: 121-135 (e90).
Development and gastrulation in Hyloxalus vertebral is and
Dendrobates auratus (Anura: Dendrobatidae)
Francisca Hervas, Karina P. Torres, Paola Montenegro-Larrea, and ^Eugenia M. del Pino
Escuela de Ciencias Bioldgicas, Pontificia Universidad Catolica del Ecuador, Av. 12 de Octubre 1076 y Roca, Quito 170517, ECUADOR
Abstract. — ^We document the embryonic development of Hyloxalus vertebralis, a frog species of
the Ecuadorian highlands, declared as Critically Endangered by the International Union for the
Conservation of Nature (lUCN) due to significant declines in its populations. Our work may be of
value for conservation and management of this endangered frog, especially as it is being bred in
captivity to ensure against extinction. We were able to analyze and compare the development of H.
vertebralis with Dendrobates auratus (Dendrobatidae), and other frogs, because of the successful
reproduction in captivity of Ecuadorian frogs at the Balsa de los Sapos, Centre of Amphibian
Investigation and Conservation (CICA), of the Pontificia Universidad Catolica del Ecuador, in
Quito. Embryos were fixed, and the external and internal morphology was described from whole
mounts, and serial sections. Cellular morphology was analyzed by staining nuclei. Embryos of H.
vertebralis and D. auratus developed from eggs that were 2.6 and 3.5 mm in diameter, respectively.
In spite of the large size of their eggs, the morphology of H. vertebralis embryos from cleavage to
hatching was similar to the morphology of Epipedobates machalilla (Dendrobatidae) embryos. The
comparison of gastrulation morphology was extended to six additional species of Dendrobatidae
(E. machalilla, Epipedobates anthonyi, Epipedobates tricolor, H. vertebralis, Ameerega bilinguis, D.
auratus), and to Xenopus laevis (Pipidae), and Gastrotheca riobambae (Hemiphractidae). We found
that elongation of the notochord occurs after blastopore closure in the six species of dendrobatid
frogs, as in G. riobambae; whereas gastrulation and notochord elongation overlap during X. laevis
development. We propose that the separation of gastrulation from notochord elongation may relate
to slower development patterns, probably associated with the terrestrial reproductive strategies
of dendrobatid frogs and marsupial frogs. This analysis contributes to the knowledge of frog
embryology and gastrulation, and provides developmental information that may be useful for the
conservation and management of H. vertebralis.
Key words. Ameerega bilinguis, Epipedobates machalilla, Epipedobates anthonyi, Epipedobates tricolor, notochord,
neurula
Citation: Hervas F, Torres KP, Montenegro-Larrea P, del Pino EM. 2015. Development and gastrulation in Hyloxalus vertebralis and Dendrobates
auratus (Anura: Dendrobatidae). Amphibian & Reptiie Conservation 8(1) [Special Section]: 121-135 (e90).
Copyright: © 2015 Hervas et al. This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-
NoDerivatives 4.0 International License, which permits unrestricted use for non-commercial and education purposes only, in any medium, provided
the original author and the official and authorized publication sources are recognized and properly credited. The official and authorized publication
credit sources, which will be duly enforced, are as follows: official journal title Amphibian & Reptiie Conservation; official journal website <amphibian-
reptile-conservation. org> .
Received: 14 May 2014; Accepted: 12 November 2014; Published: 22 March 2015
Introduction
We analyzed the embryonic development of Hyloxalus
vertebralis and Dendrobates auratus from cleavage to
tadpole hatching and compared these patterns with the
development of Epipedobates machalilla (Dendrobati-
dae) and Xenopus laevis (Pipidae), frogs with well-stud-
ied development. These comparisons were then extended
to embryos of other Ecuadorian, neotropical frogs (del
Pino et al. 2004, 2007; Moya et al. 2007; Nieuwkoop and
Faber 1994). Our aim was to extend the analysis of frog
embryonic development to additional species and to pro-
vide information that may be useful for the conservation
and management of H. vertebralis, an endangered frog.
Most of the H. vertebralis population has disappeared,
possibly due to chytridiomycosis infection and habitat
destruction. For these reasons, the International Union
for Conservation of Nature (lUCN) declared H. verte-
Correspondence. Email: ^edelpino@puce.edu.ec, tel: (593 2) 299 1 700 extension 1280; fax: (593 2) 299 1725.
Amphib. Reptile Conserv.
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Hervas et al.
Fig. 1. External views of H. vertebralis embryos from cleavage to the neurula stage. (A) Stage 2: Two-cell stage. (B) Stage 4: Eight-
cell stage. (C) Stage 5: Sixteen-cell stage. (D) Stage 9: Advanced blastula. (E) Stage 10: Early gastrula. (F) Stage 11: Mid-gastrula.
(G) Stage 12: Late-gastrula. (H) Stage 12.5: Late-gastrula with a small yolk plug. (I) Stage 13: Slit-blastopore stage. (J) Stage 13.5:
Advanced slit-blastopore stage. The neural plate was visible. (K) Stage 14: Early neural fold stage. (L) Stage 15: Mid-neural fold
stage. In this and the following figures, the developmental stage (st) is given in top right-hand comer of each image; b, blastopore;
c, cleavage furrow; dl, dorsal blastopore lip; np, neural plate; nt, neural tube; yp, yolk plug.
bralis as Critically Endangered (Coloma et al. 2004). It
is currently being bred in captivity to guard against ex-
tinction.
Hyloxalus vertebralis occurs at elevations of 1,770-
3,500 m above sea level in the inter-Andean valleys of
Ecuador. In addition, it occurs on the eastern and west-
ern slopes of the Andes in central and southern Ecuador,
respectively (Coloma 1995). Its habitat is the cloud for-
est and it has also been found in ponds, open areas, and
streams. The nests consist of 5-12 eggs that are placed on
the ground (Coloma 1995). After the tadpoles hatch, the
males transport them to streams for further development
(Coloma 1995).
Dendrobates auratus is distributed from southeastern
Nicaragua to northwestern Colombia (Solis et al. 2004).
This species does not occur in Ecuador. These frogs de-
posit their eggs in terrestrial nests, and embryonic de-
velopment occurs inside the egg capsules until tadpole
hatches in the leaf litter. Brood care is performed by the
male. After hatching, tadpoles are transported individu-
ally by the male to small seasonal pools (Solis et al.
2004). Eggs of D. auratus are the largest among the den-
drobatids and measure 3.5 mm in diameter (del Pino et
al. 2007; Hervas and del Pino 2013).
Dendrobatid frogs are of great developmental inter-
est because of their great variation in egg size (Table 1),
and their modified pattern of gastrulation. Notochord
elongation occurs after gastrulation in E. machalilla, and
Epipedobates anthonyi, as in the Marsupial frog, Gas-
trotheca riobambae (Hemiphractidae); whereas, the on-
set of notochord elongation is a feature of the Xenopus
laevis mid-gastrula (Benftez and del Pino 2002; Keller
and Shook 2004; del Pino et al. 2007; Moya et al. 2007;
Montenegro-Larrea and del Pino 2011; Elinson and del
Pino 2012). For this reason, we compared the gastrula-
tion characteristics of Epipedobates anthonyi, Epipe-
dobates tricolor, H. vertebralis, Ameerega bilinguis,
and D. auratus with E. machalilla (Dendrobatidae). In
a previous study, Ameerega bilinguis was identified as
Epipedobates ingeri (del Pino et al. 2007). This analysis
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Development and gastrulation in Hyloxalus vertebralis and Dendrobates auratus
H. vertebralis
Fig. 2. External views of H. vertebralis embryos from closure of the neural tube to hatching. (A) Stage 16.5: Closure of the neural
tube. (B) Stage 17: Tail bud stage. The brachial arches protruded on the sides of the head. (C) Stage 19: Embryo at the muscular
response stage. (D) Stage 19.5: Gill buds of the two external gill pairs were visible. (E) Stage 21: Development of the external
gills. There were seven branches in the first gill pair and the second gill pair was unbranched. (F) Stage 25: Embryo at hatching, hr,
branchial arch; e, eye; g, gills; gb, gill bud; tf, tail fin.
Table 1. Gastrulation in dendrobatid frogs in comparison withX laevis (Pipidae) and G. riobambae (Hemiphractidae).
Family and Species
Eggs per
clutch (No. of
clutches)
Egg diam-
eter (mm)
Gastrulation
time (hrs)®
Archenteron
elongation
Onset of noto-
chord elongation
References^
Pipidae
Xenopus laevis
1.3
5
Early gastrula'’
Mid gastrula^
1
Dendrobatidae
Epipedobates machalilla
15 (72)
1.6
65
Late gastrula“
After gastrulation®
2
Epipedobates anthonyi
18 (30)
2.0
36
Late gastrula“
After gastrulation®
3
Epipedobates tricolor
13 (34)
2.0
36
Late gastrula“
After gastrulation®
2
Hyloxalus vertebralis
13 (39)
2.6
39
Late gastrula“
After gastrulation®
4
Ameerega bilinguis
10 (04)
3.0
55
Late gastrula'*
After gastrulation®
4
Dendrobates auratus
05 (42)
3.5
72
Late gastrula'*
After gastrulation®
1
Hemiphractidae
Gastrotheca riobambae
87
3.0
168
After gastrulation®
After gastrulation®
5
‘‘Time from stages 10-13. Embryo culture temperatures for: X. laevis 23 °C, and 18-21 °C for other frogs; '’StagelO; “Stage 12.5;
‘^Stage 12; “Stage 13; ‘^Stage 11; ^References: 1, (del Pino et al. 2007); 2, (del Pino et al. 2004); 3, (Montenegro-Larrea and del Pino
2011); 4, This work; 5, (del Pino 1996; Moya et al. 2007).
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Hervas et al.
Fig. 3. Internal morphology of the H. vertebralis and D. auratus early gastrula. Micrographs of H. vertebralis embryos are shown
in A, B, E-G, and micrographs of D. auratus embryos are shown in C, D, H-J. Sections shown in E-G, I-J were stained for cell
nuclei. (A) Stage 9: Sagittal section of an advanced blastula. (B) Stage 10: Sagittal section of an early gastrula. (C) Stage 10: Sagit-
tal section of an early gastrula. (D) Stage 11: Sagittal section of the mid-gastrula. (E) Stage 10: Higher magnification of the dorsal
blastopore groove from the embryo in B. The arrow signals a bottle cell. (F) Stage 10: The blastocoel roof of an early gastrula. It
was two-three cells in thickness. (G) Stage 12.5: One cell layer in the blastocoel roof of a late-gastrula. (H) Stage 10: Higher mag-
nification from the embryo in C. The arrow signals a bottle cell. (I) Stage 10: The blastocoel roof of an early gastrula of two cells in
thickness. (J) Stage 11: The blastocoel roof of mid-gastrula with one-two cells in thickness, bl, blastocoel; bg, blastopore groove;
dl, dorsal blastopore lip; vl, ventral blastopore lip.
of gastrulation in several dendrobatids expands previous
studies (del Pino et al. 2007; Montenegro-Larrea and del
Pino 2011). The gastrulation pattern of these dendroba-
tids is similar to the pattern of E. machalilla, with the no-
tochord elongation after completion of gastrulation (del
Pino et al. 2004, 2007; Moya et al. 2007).
We report the features of development from cleavage
to tadpole hatching of H. vertebralis and from gastrula
to tadpole hatching of D. auratus. This study expands
the report on the mode of myogenesis, neurulation, and
internal features of embryos of these two dendrobatids
(Hervas and del Pino 2013). In spite of the large size of
their eggs, the external and internal morphology from
cleavage until tadpole hatching of H. vertebralis, and D.
auratus is similar to that of E. machalilla (del Pino et al.
2004 2007; Hervas and del Pino 2013). Moreover, myo-
genesis occurs by cell interdigitation, as in embryos of
other dendrobatid frogs (del Pino et al. 2007; Hervas and
del Pino 2013).
Materials and Methods
Collection sites
Adults of Hyloxalus vertebralis were collected by Per-
nando Duenas and Italo Tapia on 10 September 2008.
The locality of collection was Azuay Province, Sevilla
de Oro, in southern Ecuador at an altitude 2,418 m above
sea level. The geographic coordinates of this site are W
78.60097, S 2.63605. The permit 016-IC-FAU-DNBAP-
MA from the Ministry of the Environment, Ecuador, al-
lowed the collection and maintenance of frogs at Pon-
tificia Universidad Catblica del Ecuador (PUCE). The
Atlanta Zoo donated adults of Dendrobates auratus to
the PUCE. Adults of both species reproduced success-
fully at the Balsa de los Sapos, Centre of Amphibian
Investigation and Conservation (CICA) of PUCE. Egg
clutches were donated to the laboratory of developmental
biology for embryonic analysis.
Analysis of embryonic development
The number of eggs per egg clutch was recorded. Em-
bryos were analyzed from cleavage until tadpole hatch-
ing, and were staged according to the E. machalilla table
of stages (del Pino et al. 2004). Embryos were cultured
in humid chambers at room temperature (18-23 °C).
Procedures for fixation of embryos in Smith’s fixative,
vibratome sectioning, and the staining of sections for cell
nuclei with the fluorescent dye Hoechst 33258 (Sigma-
Aldrich, St. Louis, MO, USA) were previously described
(del Pino et al. 2004; Moya et al. 2007). Sections were
mounted in glycerol, and microscopically examined with
normal light using a Stemi SV6 stereomicroscope (Carl
Zeiss, Oberkochen, Germany) or with fluorescent op-
tics using a Z1 Axio Observer microscope (Carl Zeiss,
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Development and gastrulation in Hyloxalus vertebralis and Dendrobates auratus
H. vertebralis D. auratus
Fig. 4. Stage 14: Early neural fold stage of H. vertebralis and D. auratus embryos. Micrographs of H. vertebralis embryos are
shown in A, C, and micrographs of D. auratus embryos are shown in B, D. Sections shown in C-D were stained for cell nuclei. (A)
Lateral view of a neural fold stage embryo. (B) Dorsal view of a neural fold stage embryo. The neural plate was visible in embryos
of the two species. (C) Cross section through the region of the trunk (Reproduced from Hervas and del Pino, 2013). (D) Cross sec-
tion through the caudal region of an embryo. The notochord was visible in C and D. b, blastopore; e, endoderm; ec, ectoderm; n,
notochord; nf, neural fold; pm, paraxial mesoderm.
Oberkochen, Germany). Embryos were photographed
with Axiocam cameras and the image capture program
Axiovision (Carl Zeiss, Oberkochen, Germany). The im-
ages were edited with Adobe Photoshop CS6. Egg diam-
eter was measured in fixed embryos with the measuring
tool of the program Axiovision (Carl Zeiss, Oberkochen,
Germany).
Results and Discussion
Clutch size and developmental time
The number of eggs ranged from 2-25 eggs, with a mean
of 13 eggs per clutch in H. vertebralis, and 2-8 eggs,
with a mean of five eggs per clutch in D. auratus (Table
1). The eggs of H. vertebralis and D. auratus measured
about 2.6 and 3.5 mm in diameter, respectively (Table
1). The diameter of H. vertebralis eggs was previously
reported to be of about three nun (Coloma 1995). The
animal hemisphere of embryos was dark brown and the
vegetal hemisphere was pale-yellow in embryos of both
frogs (Eig. lA-D; not shown forZ). auratus). Egg clutch-
es of H. vertebralis required 18 days from the two-cell
stage to tadpole hatching under laboratory conditions;
whereas 19-21 days were required from fertilization to
tadpole hatching by the six species of dendrobatid frogs
(del Pino et al. 2004, 2007; Hervas and del Pino 2013).
The similarity of developmental times suggests that pa-
rental care allows slow development in all of the spe-
cies of dendrobatid frogs examined in comparison with
X. laevis.
Embryonic development of H. vertebralis and
D. auratus
The development from early cleavage until tadpole hatch-
ing of H. vertebralis and D. auratus was divided into 25
stages, according to the staging criteria for E. machalilla
(del Pino et al. 2004) given in Table 2. Micrographs of
the external and internal morphology of H. vertebralis
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H. vertebralis D. ouratus
Fig. 5. Stage 15: Mid-neural fold stage of embryos of H. vertebralis and D. auratus. Micrographs of H. vertebralis embryos are
shown in A, C, and micrographs of D. auratus embryos are shown in B, D. Sections shown in C-D were stained for cell nuclei.
(A-B) Dorsal views of embryos. The neural tube was open in embryos of both species. (C) Cross section through the caudal region.
The notochord was not detected in this caudal region (Reproduced from Hervas and del Pino, 2013). (D) Cross section through the
trunk region. The notochord was visible, e, endoderm; ec, ectoderm; n, notochord; nf neural fold; pm, paraxial mesoderm.
and D. auratus embryos illustrate these developmental
stages (Figs. l-12).Cleavage of H. vertebralis was ho-
loblastic as in E. machalilla (del Pino et al. 2004) (Fig.
1 A-C). Cleavage of D. auratus was not observed. The H.
vertebralis blastula consisted of small, pigmented cells
in the animal hemisphere; whereas, cells of the vegetal
hemisphere were larger. The blastocoel developed during
cleavage, and was large in blastula and gastrula stage em-
bryos (Fig. 3A-D). The blastocoel roof, of the two spe-
cies, was several cell diameters in thickness at stage 10
(Fig. 3F and I), and it was reduced to one cell thickness in
the late-gastrula stage of H. vertebralis (stage 12.5; Fig.
3G). Similarly the thickness of the blastocoel roof was
reduced to one or two cells in thickness in the early gas-
trula of D. auratus (stage 11; Fig. 3J). Thickness of the
blastocoel roof in the late-gastrula was not documented
for this frog species. In E. machalilla, expansion of the
blastocoel was accompanied by reduction in its thickness
until it was a monolayer of cells in the late-gastrula (del
Pino et al. 2004).
The onset of gastrulation in H. vertebralis and D. au-
ratus was marked by the presence of the dorsal blasto-
pore lip in a sub-equatorial location (Fig. IE). A field of
bottle cells was observed at the blastopore groove (Figs.
3E, H), as in X. laevis and E. machalilla (Hardin and
Keller 1988; Moya et al. 2007). The gastrula developed
a conspicuous yolk plug that became smaller during gas-
trulation, until it was totally retracted by the end of gas-
trulation (Fig. lE-I). The closed blastopore looked like a
small slit in stage 13 embryos (Fig. II), as in E. macha-
lilla and other frogs (del Pino et al. 2004). Internally, a
small dorsal archenteron developed, which did not elon-
gate until stage 13 in//, vertebralis (Fig. 12J-L), as 'mE.
machalilla (del Pino et al. 2004); whereas in D. auratus,
the archenteron was already large and inflated at stage 12
(Fig. 12P) (del Pino et al. 2007).
The neural plate developed in late stage 13 (Fig. IJ).
In stage 14, the neural folds were elevated (Figs. IK;
4A-B). The notochord was observed underneath the
neural plate of stage 14 embryos (Fig. 4C-D). The neu-
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Development and gastrulation in Hyloxalus vertebralis and Dendrobates auratus
Table 2. Stages of development of H. vertebralis and D. auratus in comparison with the E. machalilla table of development.
Stage^
Characteristics of embryos
D
X
G
1
1
1
Fertilization (not shown).
1
-
2
Gray crescent (not shown).
2
2
3
Two-cell stage (Fig. lA). This stage was observed only for //. vertebralis.
3
3
4
Four-cell stage (not shown).
4
4
5
Eight-cell stage (Fig. IB). This stage was observed only for H. vertebralis .
5
5
6
Sixteen-cell stage (Fig. 1C). This stage was observed only for H. vertebralis.
6
6
7
Thirty-two cell stage (not shown).
7
7
8
Large-cell blastula (not shown).
8
8
-
Medium-cell blastula (not shown).
9
9
9
Advanced blastula (Fig. ID; 3A).
10
10
10
Early gastrula. The dorsal blastopore lip was formed (Eig. IE), had a subequatoiial location (Eigs. 3B, C), and there
were bottle cells making the onset of cell ingression at the blastopore, as shown for both species (Eigs. 3E, H).The blas-
tocoel was a large cavity, and its roof was several cells in thickness. The thickness of the blastocoel roof was reduced to
a single cell in the late gastrula (Eigs. 3E, G, I, J).
11
11
11
Mid-gastrula with a yolk plug that measured about 1/2 of the embryo's diameter (Eig. IE). Internally, the ventral blasto-
pore lip was formed as shown for D. auratus (Eig. 3D).
12
12
12
Late gastrula with a yolk plug that was 1/3 of the embryo's diameter or smaller (Eigs. IG). The archenteron of H. verte-
bralis was smaller than D. auratus (Eigs. 12J, K, P, Q).
13
13
13
Slit blastopore stage (Eig. 11). Internally, the archenteron was elongated. A large circumblastoporal collar was visible
(Eigs. 12L; R). The neural plate became visible in the late stage 13 (Eig. IJ).
14
14
14
Early neural fold stage. Images of H. vertebralis (Eig. IK; 4A, C), and of D. auratus (Eigs. 4B, D). In the trunk region of
both species, the neural plate, notochord, and mesoderm were visible (Eigs. 4C, D).
15
16
15
Mid-neural fold stage. The neural folds approached each other. Images of H. vertebralis (Eig. IL; 5A, C), and of D. au-
ratus (Eigs. 5B, D). The neural folds were elevated and touched each other in the trunk region {H. vertebralis, Eig. 5C);
but were open in the cephalic region {D. auratus, Eig. 5D).
16
20
16
Closure of the neural tube in H. vertebralis (Eig. 2A; 6A, C), and D. auratus (Eigs. 6B, D). Closure of the neural tube
was complete in both species.
17
24
17
Tail bud stage. The tail bud and the head region protruded beyond the yolky endoderm in H. vertebralis (Eig. 2B; 7); not
shown for D. auratus. The epidermis, neural tube, notochord, somites, and endoderm were visible in the trunk region
(Eig. 7B, C).
18
26
18
Muscular activity. The branchial arches protruded on the sides of the head. The eye vesicles were small (not shown). Im-
ages of H. vertebralis (Eigs. 8A, C, E), and of D. auratus (Eigs. 8B, D, E). The epidermis, neural tube, notochord, rows
of somites, and endoderm were visible in the trunk region of both species (Eigs. 8C-E).
19
33
19
Heart beat and external gill buds. The gill buds of the two pairs of external gills were visible. Images of H. vertebralis
(Eigs. 2C, D; 9A, C, E), and of D. auratus (Figs. 9B, D, F). The dorsal fin was visible (Fig. 9C), the notochord was vacu-
olated (Fig. 9C), and the pronephros was detectable (Fig. 9D). The number of somites increased (Figs. 9E, F).
20
40
20
Circulation to the external gills. The first gill pair had four or more branches. Images ofH. vertebralis (Fig. lOA). Inter-
nally, the otocysts, brain, notochord, and somites were observed, as shown for 77. vertebralis (Fig. IOC).
21
41
21
Development of the external gills. The first pair of external gills had seven branches in H. vertebralis. The second pair of
external gills was small and unbranched (Fig. 2E). In D. auratus, the first gill pair had six branches and the second gill
pair developed two branches. The tail became elongated (not shown).
22
41
22
The external gills enlarged and eye pigment. The eyes contained pigment. Images of H. vertebralis (Fig. lOB). Inter-
nally, the otocysts, eye, internal gills, notochord, and somites were observed, as shown for 77. vertebralis (Fig. lOD).
23
43
23
The external gills reached their full size (Fig. 11). The first pair of external gills had eight and nine branches in 77 ver-
tebralis and D. auratus, respectively. The second gill pair was unbranched in 77 vertebralis and had two branches in D.
auratus (Figs. 11 A, B). The opercular fold was visible. The eyes and the body were pigmented. Internally the epidermis,
eye, otocysts, and somites were detected. Images of 77 vertebralis (Figs. 11 A, C) and of D. auratus (Figs. IIB, D).
24
44
24
The external gills were visible only on the left side. The operculum was closed on the right side (not shown).
25
45
25
The spiracle was formed. The embryos hatched and had the appearance of a tadpole. Internally, the brain, otocysts,
somites, and yolky endoderm were observed. Images of 77 vertebralis (Fig. 2F, llE-G).
'D, stages of the dendrobatid frogs, H. vertebralis and D. auratus, according to the E. machalilla standard stages of development
(del Pino et al. 2004); X, normal stages of X. laevis development (Nieuwkoop and Faber 1994); G, the generalized table of frog
development (Gosner 1960).
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H. vertebralis D. auratus
Fig. 6. Stage 16: Closure of the neural tube in embryos of H. vertebralis and D. auratus. Micrographs of H. vertebralis embryos
are shown in A, C, and micrographs of D. auratus embryos are shown in B, D. Sections shown in C-D were stained for cell nuclei.
(A-B) Dorsal views of embryos. The neural tube was closed. The branchial arches were visible in the head region. (C) Cross sec-
tion through the rostral region, anterior to the notochord. The neural tube was completely closed. (Reproduced from Hervas and del
Pino, 2013). (D) Cross sections through the trunk region of an embryo. The somites were visible, br, branchial arch; e, endoderm;
ec, ectoderm; hy, hyoid arch; ma, mandibular arch; m, mesoderm; n notochord; nt, neural tube; s, somite.
ral folds became closed along the midline during stages
15-16 (Figs.lL; 2A; 5; 6). The external and internal
characteristics of the neumla from stages 14-16 of H.
vertebralis and D. auratus were compared (Figs. 4-6)
and were found to be similar to E. machalilla embryos
(del Pino et al. 2004).
The tail bud embryos (stage 17) of H. vertebralis
were examined in their external and internal morphol-
ogy (Figs. 2B; 7). The body became elongated and the
head and tail regions protruded over the large yolky en-
doderm. The branchial arches were visible (Fig. 7A), and
the brain and neural tube were detected. Embryos of this
stage contained numerous somites (Fig. 7B, C). Embryos
of stage 18 were characterized by muscular activity, and
the embryos were longer. Buds of the external gills were
detected in the head region (Eig. 8A, B). Somites, the
notochord and neural tube were detected in the trunk
region (Eig. 8C, D). A row of somites was detected on
each side of the notochord (Eig. 8E, E). Myogenesis in
both species occurred by cell interdigitation, as in other
dendrobatid frogs and in the Marsupial frog, G. riobam-
bae\ whereas, cell rotation is the pattern for X. laevis
myogenesis (Gatherer and del Pino 1992; Hervas and del
Pino 2013). Gill buds were larger in stage 19 embryos
(Pigs. 2C-D; 9), and the external gills were fully devel-
oped in embryos of stage 22-23. The first gill pair of H.
vertebralis developed eight branches, and the second
pair was unbranched; whereas, embryos of D. auratus
developed nine and two branches in the first and second
gill pairs, respectively (Pigs. 10; 11 A, B). The number
of gill branches in the first and second pair of external
gills varies among species of Dendrobatidae (del Pino et
al. 2004). The tail became longer in embryos of stages
18-25, the brain, spinal cord, somites, and internal or-
gans developed and the embryos gradually acquired the
tadpole shape in both species (Pigs. 2C-P, 8-11). The
processes of neurulation, somitogenesis, and internal em-
bryo morphology of H. vertebralis and D. auratus were
similar to the patterns described for other species of den-
drobatid frogs (del Pino et al. 2004, 2007). Embryos of
H. vertebralis hatched at stage 25 (Pigs. 2P, llE-G). The
mouth had darkly pigmented teeth (Pig. UP), the body
had dark pigment, and the embryo had the appearance of
a tadpole (Pigs. 2P, UP, G).
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Development and gastrulation in Hyloxalus vertebralis and Dendrobates auratus
H. vertebralis
C St 17
Fig. 7. Stage 17: Tail bud stage of H. vertebralis embryos. (A)
Lateral view of an embryo. (B) Cross section through the trunk
region of the embryo in A. (C) Horizontal section at the level
of the notochord and somites with the rostral region towards
the left. A row of somites was detected on each side of the no-
tochord. A portion of the neural tube was detected in the rostral
region of the section, hr, branchial arch; e, endoderm; ec, ec-
toderm; hy, hyoid arch; ma, mandibular arch; n notochord; nt,
neural tube; s, somite.
Comparative analysis of gastrulation
Gastrulation is characterized by common morphogenet-
ic events that occur in all of the analyzed frog species.
Formation of the dorsal blastopore lip, its development
to enclose a yolk plug, and the process of internaliza-
tion of cells at the blastopore lip by the movements of
involution are among these common morphogenetic pro-
cesses (Elinson and del Pino 2012). Other developmental
events, however, may be dissociated from gastrulation
in some frog species. In particular, dorsal convergence
and extension and the onset of notochord elongation
are separated from gastrulation in the Marsupial frog,
G. riobambae, and in dendrobatid frogs; whereas, these
events occur simultaneously with gastrulation in X. lae-
vis and in Engystomops (Leptodactylidae) (Table 1) (del
Pino 1996; Benitez and del Pino 2002; Keller and Shook
2004; Moya et al. 2007; Elinson and del Pino 2012).
The simultaneous occurrence of gastrulation and on-
set of notochord elongation may be related to the repro-
ductive adaptation of frogs for rapid development under
unstable environmental conditions such as the aquatic
environment in which embryos of X. laevis develop,
or the development in floating foam nests in species of
Engystomops (Elinson and del Pino 2012). Embryos of
these frogs require from 5 hours to 12.5 hours from the
onset of gastrulation to blastopore closure (Stage 10-13)
(Nieuwkoop and Eaber 1994; Romero-Carvajal et al.
2009). Elongation of the notochord and gastrulation oc-
cur simultaneously in embryos ofX. laevis, Engystomops
coloradorum, and Engystomops randi (Leptodactylidae)
(Keller and Shook 2004; Romero-Carvajal et al. 2009;
Venegas-Eerrm et al. 2010). Early elongation of the no-
tochord may be required for embryos to rapidly acquire
the elongated tadpole shape in the unstable conditions of
their reproductive environments.
The most divergent mode of gastrulation was detected
in embryos of the Marsupial frog, G. riobambae. Gas-
trulation results in the formation of an embryonic disk
from which the body of the embryo develops (del Pino
and Elinson 1983). Cells that involute during gastrulation
accumulate in the blastopore lip, and after blastopore clo-
sure give rise to an embryonic disk of small cells, visible
on the surface. Internally, the small cells that involuted
during gastrulation accumulated in the embryonic disk
and in its internal circumblastoporal collar (Moya et al.
2007). Eormation of the embryonic disk of G. riobambae
is associated with delayed onset of notochord elongation
that only starts once the blastopore is closed (del Pino
1996). Embryos of the Marsupial frog, G. riobambae de-
velop slowly, and take a total of 168 hours from the onset
of gastrulation to its completion (Table 1).
As in G. riobambae, cells that involuted during gas-
trulation became accumulated in a large circumblasto-
poral collar in embryos of dendrobatid frogs, with sepa-
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H. vertebrolis D. auratus
Fig. 8. Stage 18: Muscular activity stage of H. vertebmlis and D. auratus embryos. Micrographs of H. vertebralis embryos are
shown in A, C, E, and micrographs of D. auratus embryos are shown in B, D, F. Sections shown in C-D were stained for cell nuclei.
(A) Lateral view of an embryo. (B) Dorsal view of an embryo. The gill buds were visible on each side of the head in embryos of
both species. (C-D) Cross sections through the trunk. The cavity in D corresponds to the gut. (E-F) Horizontal sections. A row of
numerous somites was detected on each side of the notochord. The brain and the otocysts were visible in E, and the gut was visible
in F. bn, brain; ec, ectoderm; g, gut; gb, gill bud; hy, hyoid arch; ma, mandibular arch; n notochord; nt, neural tube; ot, otocyst; p,
pronephros; s, somite.
ration of the morphogenetic events of gastmlation and
the onset of notochord elongation. However, dendrobatid
frogs do not develop an embryonic disk (Elinson and del
Pino 2012). Egg size varied from 1.6 to 3.5 mm in diam-
eter among dendrobatid frogs (Table 1), and their devel-
opment was slow. Embryos of dendrobatid frogs require
36-72 hours from the onset of gastmlation to its comple-
tion (Stage 10-13; Table 1). We analyzed the characteris-
tics of the gastmla in dendrobatid embryos derived from
eggs of different diameters (Table 1; Eig. 12). Protection
of embryos in the terrestrial nests of dendrobatids or
inside a pouch of the mother in G. riobambae may al-
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Development and gastrulation in Hyloxalus vertebralis and Dendrobates auratus
H. vertebralis D. auratus
Fig. 9. Stage 19: Muscular response stage of H. vertebralis and D. auratus embryos. Micrographs of H. vertebralis embryos are
shown in A, C, E, and micrographs of D. auratus embryos are shown in B, D, F. (A) Lateral view of an embryo. (B) Dorsal view
of an embryo. The developing gills were visible. (C-D) Cross sections through the trunk. The dorsal fin was visible in C, and the
pronephros in D. (E) Horizontal section at the level of the gut. (F) Horizontal section at the level of the brain and the gut. bn, brain;
df, dorsal fin; fg, first gill pair; g, gut; gb, gill bud; hy, hyoid arch; ma, mandibular arch; n notochord; nt, neural tube; p, pronephros;
s, somite.
low slow development and the separation of gastrulation
from notochord elongation (Elinson and del Pino 2012).
Details of the morphology of the H. vertebralis and D.
auratus gastrula are illustrated in Fig. lE-I, Fig. 3B-J,
and Fig. 12J-F, P-R. The archenteron roof remained
relatively thin during gastrulation in H. vertebralis and
D. auratus in comparison with stage 13 embryos of X.
laevis (Fig. 12C, J-L, P-R). Elongation and inflation of
the archenteron varied greatly among dendrobatids. The
archenteron remained small during gastrulation and be-
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Hervas et al.
H. vertebralis
Fig. 10. Stages 20-22: External gill development in H. vertebralis. (A) Stage 20: Circulation to the external gills. Three branches
were visible in the first gill pair. The second gill pair was unbranched. (B) Stage 22: The external gills enlarged. Seven branches
were visible in the first gill pair. The second gill pair was unbranched. (C) Stage 20: Horizontal section at the level of the brain. (D)
Stage 22: Horizontal section at the level of the internal gills, bn, brain; ey, eye; ec, ectoderm; fg, first gill pair; g, internal gill; nt,
notochord; ot, otocyst; s, somite; sg, second gill pair.
came elongated and inflated after gastmlation in H. ver-
tebralis (Fig. 12J-L); whereas, at stage 12, the archen-
teron was already elongated in the very large embryos
of A. bilinguis and D. auratus (Fig. 12 M, P). Moreover
inflation of the archenteron was already deteeted in stage
12 embryos of D. auratus (Fig. 12 P). In other species of
dendrobatids, we detected variation in the level of arch-
enteron elongation and inflation (Fig. 12D-R). We con-
cluded that in A. bilinguis, and D. auratus, dendrobatids
with very large eggs, the elongation of the archenteron
begins earlier in comparison with embryos of dendroba-
tid frogs with smaller eggs such E. machalilla (Table 1)
(del Pino et al. 2007).
In spite of the differences detected in the onset of
archenteron elongation, the cells that involuted during
gastmlation became accumulated in a large circumblas-
toporal collar at stage 13 in all of the dendrobatid frogs
analyzed, as previously reported for E. machalilla, and
shown for E. anthonyi, E. tricolor, H. vertebralis, A. bi-
linguis, andZ). auratus, (Fig. 12F, I, L, O, R) (Moya et al.
2007). Notochord elongation is dissociated from gastm-
lation in these frogs (Bemtez and del Pino 2002; del Pino
et al. 2007; Moya et al. 2007; Venegas-Ferrfn et al. 2010;
Montenegro-Larrea and del Pino 2011).
The comparative analysis of gastmlation indicates
that in spite of the great variation in egg size and onset of
Amphib. Reptile Conserv. 132 March 2015 | Volume 8 | Number 1 | e90
arehenteron elongation and inflation, the Dendrobatidae
species examined develop a large circumblastoporal col-
lar as a result of gastmlation (Fig. 12D-R; Table 1) (del
Pino et al. 2007; Moya et al. 2007; Montenegro-Larrea
and del Pino 2011). Moreover, notochord elongation is
delayed until after blastopore closure as in G. riobambae.
In spite of their large circumblastoporal collar, dendro-
batid frog embryos did not develop an embryonic disk.
Conclusions
Development of the dendrobatid frogs, H. vertebralis
and D. auratus, shared the developmental characteristics
described for E. machalilla (del Pino et al. 2004). Gas-
tmlation and notochord elongation occurred as separate
morphogenetic events in these frogs in comparison with
additional species of Dendrobatidae. Development in a
somewhat stable terrestrial environment may be associat-
ed with the separation of these developmental events and
with comparatively slow development. The developmen-
tal analysis of H. vertebralis and other frogs contributes
to a better knowledge of their biology and may contribute
to the conservation and reproductive management of en-
dangered frogs.
Development and gastrulation in Hyloxalus vertebralis and Dendrobates auratus
H. vertebralis
D. auratus
velopmental Biology of PUCE for their assistance. In
particular, we acknowledge the help of Alexandra Vargas
with the preparation of illustrations. We thank Clifford
Keil for valuable criticism and for language revision.
This study received the support of research grants from
PUCE.
Fig. 11. Stages 23-25: Complete development of the external gills
to tadpole hatching in embryos of H. vertebralis and D. auratus.
Micrographs of H. vertebralis embryos are shown in A, C, E, F,
G, and micrographs of D. auratus embryos are shown in B, D.
(A) Stage 23 of H. vertebralis: Full development of external gills.
The first gill pair of the external gills had eight branches, which
at this stage were fully extended. The second gill pair of external
gills was unbranched. (B) Stage 23 of D. auratus: The first gill
pair of the external gills had nine branches, which at this stage
were fully extended. The second pair of external gills was smaller
and had two branches. In embryos of both species the eyes and the
body were pigmented. Tbe tail was elongated. (C) Stage 23: Sag-
ittal section. The section was done through the embrionary brain
and somites. The eyes, notochord, and tail fin were observed. (D)
Stage 23: Horizontal section at tbe level of somites. The eyes and
otocysts were visible. (E) Stage 25: Head of a tadpole at hatching
in dorsal view. The eyes were visible. (F) Stage 25: Ventral view
of the head of the tadpole shown in A. The spiracle was visible.
(G) Stage 25: Horizontal section of a tadpole at hatching at the
level of the otocysts. The eyes, otocysts, and somites were visible,
bn, brain; ey, eye; ep, epidermis, fg, first gill pair; n, notochord;
ot, otocyst; sg, second gill pair; s, somite; tf, tail fin; y, yolky en-
doderm.
Acknowledgments. — We express our thanks to the Cen-
tre of Amphibian Investigation and Conservation (CICA),
Balsa de los Sapos of the Pontificia Universidad Catolica
del Ecuador (PUCE) for the donation of embryos. We ex-
press gratitude to the members of the Laboratory of De-
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St 12 St 12,5 St 13
Fig. 12. Gastrulation of dendrobatid frogs in comparison withX
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column headings: Stage 12, late-gastrula; Stage 12.5, advanced
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Development and gastrulation in Hyloxalus vertebralis and Dendrobates auratus
Francisca Hervas was Adjunct Professor and developmental biology researcher at the School of Biologi-
cal Sciences, Pontificia Universidad Catolica del Ecuador (PUCE, 2014), in Quito. She holds a Eicencia-
tura in Biological Sciences from PUCE, and is enrolled in the PUCE master's degree program in conserva-
tion biology. Her Licenciatura thesis is the study of the morphology of the neurula and more advanced
embryos of the species Hyloxalus vertebralis and Dendrobates auratus', she also analyzed the mode of
myogenesis in the large embryos of these frogs. Her research interests are focused on amphibians, with an
emphasis on Ecuadorian species.
Karina P. Torres is a graduate of the Eicenciatura in Biological Sciences Program at the Pontificia Uni-
versidad Catolica del Ecuador (PUCE) in Quito (2014). For her thesis research she investigated the early
development of Hyloxalus vertebralis (Dendrobatidae) in the Eaboratory of Developmental Biology at
PUCE. Her research centers in the analysis of the morphological characteristics of the H. vertebralis gas-
trula in comparison with other dendrobatid frogs.
Paola Montenegro-Larrea is a Ph.D. student at the Interdisciplinary Fife Sciences, Purdue University,
West Lafayette, Indiana, USA. She holds a M.S. in molecular genetics and diagnostics from The University
of Nottingham, United Kingdom, and a Licenciatura in biology from the Pontificia Universidad Catolica
del Ecuador (PUCE), in Quito. Her Licenciatura thesis researched the characterization of gastrula mor-
phology in four Ecuadorian species of Dendrobatid frogs with eggs of different sizes. Earlier in her career,
she took part in the establishment of the Molecular Genetics Laboratory at the hospital of the Ecuadorian
Armed Forces in Quito (Hospital de las Fuerzas Armadas del Ecuador).
Eugenia M. del Pino is professor of biological sciences (retired) at the Pontificia Universidad Catolica del
Ecuador (PUCE) in Quito. She studied the reproduction and development of marsupial frogs (Hemiphrac-
tidae) in comparison with Xenopus laevis, the model organism of frog developmental biology and with
several frogs from Ecuador. Her studies are done in collaboration with PUCE students. Her analyses of
development reveal important variation in morphology and developmental time among frogs. The devel-
opmental data is significant for the comparative analysis of frog early embryonic development, and provide
base line information about the biology of several frog species.
Amphib. Reptile Conserv.
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March 2015 I Volume 8 | Number 1 | e90
Official journal website:
amphibian-reptile-conservation.org
Amphibian & Reptiie Conservation
8(1) [Special Section]: 136-140 (e91).
Short Communication
First records of Anolis ventrimaculatus Boulenger, 1911
(Squamata: Iguanidae) in Ecuador
Ternando Ayala- Varela, ^Julian A. Velasco, ^Martha Calderon-Espinosa, "^Alejandro F. Arteaga,
^’ Yerka Sagredo, and ^ ’^Sebastian Valverde
^Escuela de Ciencias Bioldgicas, Pontificia Universidad Catolica del Ecuador, Avenida 12 de Octubre 1076 y Roca, Apartado 17-01-2184, Quito,
ECUADOR ^Laboratorio de Andlisis Espaciales, Instituto de Biologia, Universidad Nacional Autonoma de Mexico, MEXICO D.F ^Instituto de
Ciencias Naturales, Edificio 425, oficina 111, Universidad Nacional de Colombia, Sede Bogota, COLOMBIA "^Tropical Herping, Av Eloy Alfaro
N39-202 y Jose Puerta. Ed Montecatini. Quito, ECUADOR
Abstract— \Ne report the first records of Anoiis ventrimaculatus for Ecuador based on twelve
specimens from three localities: Chical (Provincia Carchi), El Cristal (Provincia Esmeraldas), and
Lita (Provincia Imbabura). The locality in the Provincia Carchi lies approximately 16 km S from the
nearest record (Nambi, Department Nariho, Colombia). We also present information on scalation
and coloration.
Key words. Anole lizards, color, distribution, Ecuador, scalation
Citation: Ayala- Varela F, Velasco JA, Calderon-Espinosa M, Arteaga AF, Sagredo Y, Valverde S. 2015. First records of Anolis ventrimaculatus Bou-
lenger, 1911 (Squamata: Iguanidae) in Ecuador. Amphibian & Reptile Conservation 80) [Special Section]: 136-140 (e91).
Copyright: © 2015 Ayala- Varela et al. This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommer-
cialNoDerivatives 4.0 International License, which permits unrestricted use for non-commercial and education purposes only, in any medium, provided
the original author and the official and authorized publication sources are recognized and properly credited. The official and authorized publication
credit sources, which will be duly enforced, are as follows: official journal title Amphibian & Reptile Conservation] official journal website <amphibian-
reptile-conservation. org>.
Received: 13 December 2014; Accepted: 02 March 2015; Published: 04 April 2015.
Thirty-seven species of Anolis have been reported for-
mally from Ecuador (Ayala- Varela et al. 2014). Anolis
ventrimaculatus Boulenger 1911 was described from two
syntypes, an adult female and a juvenile from Rio San
Juan, Department Risaralda, Colombia. Williams and
Duellman (1984) designated the adult female syntype
(BMNH 1946.8.13.5) as the lectotype.
Anolis ventrimaculatus is assigned to the aequatoria-
lis series Castaneda and de Queiroz (2013) by having a
moderate to large body size, narrow toe lamellae, small
head scales, smooth ventral scales, and uniform dorsal
scalation. It is assigned to the eulaemus-subgroup based
on a typical Anolis digit, in which the distal lamellae of
phalanx III distinctly overlap the first proximal subdigital
scale of phalanx II (Williams 1976; Williams and Du-
ellman 1984; Castaneda and de Queiroz 2013).
Eleven species of the eulaemus-subgroup occur on
both sides of the Andes {Anolis anoriensis Velasco et al.
2010, A. antioquiae Williams 1985, A. eulaemus Bou-
lenger 1908, A. fitchi Williams and Duellman 1984, A.
gemmosus O’Shaughnessy 1875, A. maculigula Wil-
liams 1984, A. megalopithecus Rueda-Almonacid 1989,
A. otongae Ayala- Varela and Velasco 2010, A. podocar-
pus Ayala- Varela and Torres-Carvajal 2010, A. pod Aya-
la- Varela et al. 2014, and A. ventrimaculatus Boulenger
1911).
Specimens examined for comparisons are housed in
the herpetological collections of the Museo de Zoologfa,
Pontificia Universidad Catdlica del Ecuador, Quito, Ec-
uador (QCAZ); Museo de Herpetologia de la Universidad
de Antioquia, Antioquia, Colombia (MHUA); Coleccion
de Herpetologia, Universidad del Valle, Santiago de Cali,
Colombia (UVC); and Instituto de Ciencias Naturales,
Universidad Nacional de Colombia, Bogota, Colombia
(ICN). External character terminology follows Williams
et al. (1995) and Poe and Yanez-Miranda (2008). Lamel-
lar number was counted using the method of Williams
et al. (1995), i.e., only on phalanges III and IV of the
Correspondence. Email: fpayala2000@gmail.com (Corresponding author), duvelas@gmail.com, ^mlcalderone@unal.edu.co,
*yevasanu@gmail. com, fycus_8 7@hotmail. es
April 2015 | Volume 8 | Number 1 | e91
Amphib. Reptile Conserv.
136
Ayala- Varela et al.
fourth toe. Measurements were made with digital cali-
pers on preserved specimens and are given in millimeters
(mm), usually to the nearest 0.1 nun. Snout- vent length
(SVL) was measured from tip of snout to anterior edge
of cloaca. Femoral length was measured from midline
of venter to knee, with limb bent at a 90-degree angle.
Tail length was measured from anterior edge of cloaca
to distal point.
Herein we report the first records of Anolis ventrimac-
ulatus (Fig. 1) for Ecuador based on specimens collected
at three localities. Four specimens (QCAZ 3284-3286,
8934) were collected on 16 September 1992 in Lita
(0.87°, -78.45°), Provincia Imbabura; four specimens
(QCAZ 2666, 3923, 3924, 3929) were collected on Au-
gust 1994 in El Cristal (0.83°, -78.49°, 1,200-1,250 m),
Reserva Ecolbgica Cotacachi-Cayapas, Provincia Es-
meraldas; and four specimens (QCAZ 4376, 4378, 4389,
4390) were collected on 03 July 2011 in Rfo San Pablo,
near Chical (0.90°, -78.16°, 1,399-1,451 m), Provincia
Carchi. The last locality lies approximately 16 km S from
the nearest record (ICN 11981-85, 11987-989, 12097,
Nambf, Barbacoas municipality. Department Narino,
Colombia) (Pig. 1, Table 1).
The individuals from Chical (Provincia Carchi) were
captured in secondary forest; all individuals were found
on leaves, branches, or ferns from 50-150 cm above
ground; a male were found head-down, while two fe-
males were found head-down and head-up. The small-
est specimen (QCAZ 8934, juvenile, SVL = 31.4 mm)
was collected on 16 September 1992. An adult female
(QCAZ 4378) collected in July 2011 deposited one white
egg (17.11 mm x 6.44 mm). Our collections of Anolis
ventrimaculatus in Ecuador were found from 1,200 to
1,451 m above sea level. In Ecuador, this species occurs
in sympatry with A. aequatorialis, A. gemmosus, and A.
maculiventris in Chical (Provincia Carchi); with A. lyn-
chi, A. maculiventris, and A. princeps (pers. obs. Sebas-
tian Valverde) in Lita (Provincia Imbabura), and with A.
lynchi in El Cristal (Provincia Esmeraldas).
Scalation and morphometric characters of Anolis ven-
trimaculatus are presented in Table 2. Scale counts are
similar between Ecuadorian and Colombian specimens.
Our Ecuadorian specimens of Anolis ventrimaculatus are
smaller than those from Colombia (maximum SVL 62
mm and 80 mm, respectively).
Coloration in life of specimens from Ecuador was re-
corded as follows:
Adult female (QCAZ 4390, Pigs. 2 A, B): dorsal sur-
faces of head, body and tail dark brown; dorsal surface
of body with a pale brown longitudinal stripe extending
from occipital region to base of tail; limbs pale brown
with dark brown reticulation; tail pale brown; lateral sur-
face of head with two stripes, one dark brown and ex-
tending posteriorly from loreal region, through subocu-
lar region, above tympanum to level of the hind limb,
the other stripe is pale green and extending posteriorly
from loreal region, through subocular region, above tym-
Fig. 1. Distribution of Anolis ventrimaculatus in South America
(locality numbers are listed in Table 1).
panum to level of neck; lateral surface of body brown
anteriorly and olive-green near inguinal region; ventral
surface of head yellowish green with pale brown reticu-
lations; ventral surface of body cream; ventral surface of
limbs dark cream with dark brown reticulations; ventral
surface of tail dark cream.
Adult female QCAZ 4378 (Pigs. 2 C, D) differs from
the previous pattern in having the dorsal surface of body
brown, with seven dark brown blotches arranged longi-
tudinally along the midline.
Adult male (QCAZ 4389, Pigs. 2 E, P, G): When
stressed, the background of head, body, limbs and tail
was yellowish brown; dorsal surface of the neck with
two dark brown bands; dorsal surface of body with nine
dark brown blotches arranged longitudinally; limbs with
dark brown bands; dorsal surface of tail with dark brown
transversal bands, and with three dark brown blotches in
the proximal part of tail; lateral surface of head with a
darker brown first stripe, extending posteriorly from lo-
real region, through subocular region, above the tympa-
num and bifurcating into branches that continue on nu-
chal crest and shoulder, respectively; a yellowish-green
second stripe, extending posteriorly from loreal region,
through subocular region, above the tympanum to the
shoulder; black ocelli with white centers on the shoul-
der; lateral surface of body with reddish-brown bands
oriented ventroposteriorly; ventral surface of head yel-
lowish green with pale brown reticulations; ventral sur-
face of neck pale green; ventral surface of body cream;
ventral surface of limbs pale brown with dark brown re-
April 2015 I Volume 8 | Number 1 | e91
Amphib. Reptile Conserv.
137
First records of Anolis ventrimaculatus in Ecuador
Table 1. Localities of Anolis ventrimaculatus in Ecuador and Colombia.
Site number
Country
Locality
Latitude
Longitude
Source
1
Colombia
Bosque de San Antonio, km 18 via Cali-Bue-
naventura, Valle del Cauca
3.22
-76.65
JAV pers. obs
2
Colombia
Bosque de San Antonio, km 18 via Cali-Bue-
naventura, Valle del Cauca
3.51
-76.62
UVC 9737, 9749, 9896,
MHUA 1671-79, JAV pers.
obs.
3
Colombia
Antena, Cerro La Horqueta, ca. 28 km de Cali,
Valle del Cauca
3.44
-76.52
JAV pers. Obs, ICN 3567
4
Colombia
Vereda La Tulia, Mpio. Bolivar, Valle del
Cauca
4.42
-76.24
JAV pers. obs
5
Colombia
Vereda Chicoral, La Cumbre, Valle del Cauca
3.58
-76.58
JAV pers. obs, UVC 10223
6
Colombia
PRN Barbas-Bremen, Mpio. Eilandia, Quindio
4.71
-75.64
JAV pers. obs
7
Colombia
Alrededores Lago Calima, Mpio. Darien, Valle
del Cauca
3.86
-76.56
JAV pers. obs; UVC 5189-96,
ICN 3553-54
8
Colombia
Reserva Eaunistica Bosque de Yotoco, Valle
del Cauca
3.88
-76.44
JAV pers. obs
9
Colombia
Penas Blancas, Pichinde, Valle del Cauca
3.42
-76.66
UVC 223, 224
10
Colombia
Alto de Galapagos, carretera Cartago-San Jose
del Palmar, Limite Valle-Choco
4.86
-76.22
UVC 9366, UVC 8489-95
11
Colombia
PMN Arrayanal, Mpio. Apia, Risaralda
5.29
-75.90
JAV pers. obs
12
Colombia
PMN Planes de San Rafael, Mpio. Santuario,
Risaralda
5.13
-76.00
JAV pers. obs
13
Colombia
PMN Agualinda, Mpio. Mistrato, Risaralda
5.12
-75.94
JAV pers. obs
14
Colombia
PMN Verdum, vereda La Secreta, Risaralda
5.01
-76.03
JAV pers. obs
15
Colombia
Vereda Buenos Aires, Cuenca Rio Barbo,
Pereira, Risaralda
4.73
-75.58
JAV pers. obs
16
Colombia
Rio Nambi, Narino
1.30
-78.08
JAV pers. obs
17
Colombia
Reserva La Planada, Narino
1.08
-77.88
JAV pers. obs
18
Colombia
Nambi, Narino
1.02
-78.07
ICN 11981-85, 11987-889,
12097
19
Ecuador
Lita, Imbabura
0.87
-78.45
QCAZ
20
Ecuador
El Cristal, Reserva Ecol6gica Cotacachi Cay-
apas, Esmeraldas
0.83
-78.49
QCAZ
21
Ecuador
Rio San Pablo, cerca de Chical, Carchi
0.90
-78.16
QCAZ
ticulations; ventral surface of tail pale brown with small
dark brown reticulations; dewlap skin yellowish brown;
gorgetals pale green; marginals and stemals yellowish
green; iris dark brown with yellowish-brown inner ring.
The coloration of populations of Anolis ventrimacula-
tus from Colombia display a dorsal surface of the body
that is bright emerald green, or greenish-brown with
slight darker oblique bars and yellow spots on each side
of the dorsal midline; yellow spots fuse forming a series
of saddle-shaped bars that cross the back and tail, more
visible in the stressed phase. At their stressed phase, dor-
sal and lateral surface of body brown with tiny yellow
spots; lateral surface of head with a yellow line under the
eye and with a prominent pale yellow or green line over
the lips extending back over the ear opening and along
the sides of the neck; ventral surface of head yellow-
green, sometimes with reticulations; ventral surface of
body cream to yellow-green, with dark brown spots on
the sides; ventral surface of tail orange in male adults.
Some females have a dorsal surface of body with a tan
longitudinal stripe and dark edges.
Anolis ventrimaculatus has a wide range of distri-
bution, approximately 570 km in airline between the
northern and southernmost localities. However, there is
a huge distributional gap between central and southern
Colombian populations (approximately 265 km airline
between Bosque de San Antonio, Department Valle del
Cauca and the Rio Nambi, Department Narino). One of
the main reasons for this gap is the lack of extensive her-
petological inventories in these areas, particularly in both
foothills of the Andes cordilleras. More sampling effort
should addressed to these areas with the aim to fill distri-
butional gaps in several species, including Anolis lizards.
Acknowledgments. — We thank Omar Torres-Carva-
jal of the Museo de Zoologfa (QCAZ), Vivian Paez of the
Museo Herpetolbgico de Antioquia (MHUA), and John
Lynch of the Institute Nacional de Ciencias Naturales
(ICN) for the loan of museum specimens, information
about localities, and work space; Melissa Rodriguez for
helping with the map; O. Torres-Carvajal for critical and
valuable comments throughout the development of this
April 2015 | Volume 8 | Number 1 | e91
Amphib. Reptile Conserv.
138
Ayala- Varela et al.
Fig. 2. Anolis ventrimaculatus from Ecuador: female adult
(A-B, QCAZ 4390) in dorsal and ventral view, female adult
(C-D, QCAZ 4378) in dorsal and ventral view, male adult
(E-F, QCAZ 4389) in dorsal and ventral view, male dewlap (G,
QCAZ 4389) in lateral view. Photographs by F Ayala-Varela.
manuscript. This work was funded by Secretaria de Edu-
cacion Superior, Ciencia, Tecnologia e Innovacion del
Ecuador (SENESCYT). Ecuadorian specimens were col-
lected under collection permit 008-09 IC-FAU-DNB/MA
issued by Ministerio del Ambiente and were deposited
at Museo de Zoologia (QCAZ), Pontificia Universidad
Catolica del Ecuador. Julian Velasco thanks the Wildlife
Conservation Society for the support of fieldwork in Co-
lombia under permits provided by CARDER (resolution
1085 of April, 6 2010)..
Literature Cited
Ayala-Varela E, Velasco JA. 2010. A new species of dac-
tyloid anole (Squamata: Iguanidae) from the western
Andes of Ecuador. Zootaxa 2577: 46-56.
Ayala-Varela EP, Troya-Rodrfguez D, Talero-Rodrfguez
X, Torres-Carvajal O. 2014. A new Andean anole
species of the Dactyloa clade (Squamata: Iguanidae)
from western Ecuador. Amphibian & Reptile Conser-
vation 8 [Special Section]: 8-24.
Ayala-Varela EP, Torres-Carvajal O. 2010. Anew species
of dactyloid anole (Iguanidae, Polychrotinae, Anolis)
from the southeastern slopes of the Andes of Ecuador.
ZooKeys 53: 59-73.
Castaneda MR, de Queiroz K. 2013. Phylogeny of the
Dactyloa clade of Anolis lizards: new insights from
combining morphological and molecular data. Bulle-
tin of the Museum of Comparative Zoology 160(7):
345-398.
April 2015 | Volume 8 | Number 1 | e91
Amphib. Reptile Conserv.
139
First records of Anolis ventrimaculatus in Ecuador
Table 2. Scale count and measurements (mm) of specimens of Anolis ventrimaculatus from Ecuador and Colombia. Range (sample
size) and mean. SVL = snout- vent length.
Ecuador
QCAZ
Colombia
UVC, ICN, MHUA
Colombia
Williams et al. 1995
Number of scales between second canthals
14-17(10) 15.7
12-17(18) 14.6
11-21 (20)
Number of scales bordering rostral
5-7 (10) 6.4
6-8 (18) 6.8
6-10 (20)
Number of scales between supraorbital semicircles
2-5 (10) 3.6
4-6 (18) 4.8
2-6 (20)
Number of scales between interparietal and supraor-
bital semicircles
7-11 (8) 6.9
6-11 (18)8.0
5-16 (20)
Interparietal
+/-
+/very small
(+/?)
Number of loreal rows
5-8 (9) 7.9
7-9 (18) 8.2
7-11 (20)
Number of supralabials to center of eye
6-8 (10) 7.2
6-8(18) 7.4
6-8 (20)
Number of postmentals
6-8 (9) 6.3
6-9(18) 6.6
4-8 (20)
Number of sublabials in contact with infralabials
0-2 (9) 0.5
1-3 (18) 2.5
0-2 (20)
Lamellar number
16-18(10) 17.2
17-22 (18) 19.4
16-22 (20)
Number of middorsals in 5% SVL
12-15(10) 12.9
14-19(18) 16.6
-
Number of midventrals in 5% SVL
7-11 (10) 9.2
9-14(18) 11.9
-
Femur length
16.0-20.6(10) 18.5
15.4-23.1 (16) 18.9
-
Maximum SVL (male/female)
62/57
75/69
80/62
Poe S, Yanez-Miranda C. 2008. Another new species
of green Anolis (Squamata: Iguania) from the East-
ern Anders of Peru. Journal of Herpetology 42 (3):
564-571.
Williams EE. 1976 South American anoles: The species
groups. Papeis Avulsos de Zoologia 29: 259-268.
Williams EE, Duellman WE. 1984. Anolis fitchi, a new
species of the Anolis aequatorialis group from Ecua-
dor and Colombia. University of Kansas Publications,
Museum of Natural History 10: 257-266.
Williams EE, Rand H, Rand AS, O’Hara RJ. 1995. A
computer approach to the comparison and identifica-
tion of species in difficult taxonomic groups. Breviora
502: 1-47.
Fernando Ay ala- Varela is the director of the herpetology eolleetion at the Pontifieia Universidad Catdliea del
Eeuador in Quito. He reeeived his diploma at the Pontifieia Universidad Catdliea del Eeuador, Quito in 2004. He
has been interested in herpetology since childhood and has dedicated a lot of time studying the lizards of Ecuador,
specifically the taxonomy and eeology of Anolis species. His current research interests include reproductive biology
and ecology of lizards and snakes in Ecuador.
Julian A. Velasco is a Ph.D. student at Instituto de Biologia, Universidad Naeional Autdnoma de Mexieo. His doe-
toral research is focused on understanding the ecological and evolutionary processes responsible for species richness
and diversifieation of Anolis lizards. He addresses several evolutionary and ecological topics using a combination
of conceptual and methodological approaches as niche modeling, geospatial analysis, historical biogeography, and
macroecology.
Martha Calderon is the eurator of the reptile eolleetion at the Instituto de Cieneias Naturales, Universidad Na-
eional, Colombia. She obtained her doetor degree at the Universidad Naeional Autdnoma de Mexieo (UNAM) in
Mexico City. She works on ecomorphology, thermal ecology, reproductive biology, and molecular systematics of
lizards. More information can be found here: www.biodiversidadysistematicamolecular.blogspot.com
Alejandro Arteaga is a wildlife photographer and undergraduate biology student from Venezuela. In 2009, he
co-founded Tropical Herping, an institution striving to preserve tropical reptiles and amphibians through tourism,
photography, research, and education. Alejandro is author of The Amphibians and Reptiles of Mindo and several
seientifie artieles. He has deseribed three speeies new to seienee and his photographie work has been featured in
National Geographic, Anima Mundi, and the Discovery Channel.
Yerka Sagredo Nunez is an Assoeiate Researeher at the Museum of Zoology, Pontifieal Catholie University, Ee-
uador. Her baehelor’s degree was obtained in biologieal seienees from the Central University of Eeuador. Currently
she is working as an assistant in the herpetology eolleetion at the Museo de Zoologia of the Pontifieia Universidad
Catdliea del Eeuador (QCAZ). She is interesting in eeology, behavior, and taxonomy of amphibians and reptiles. She
is also involved in studies of the genus Pristimantis.
Sebastian Valverde is an Assoeiate Researeher at the Museum of Zoology at the Pontifieal Catholie University,
Ecuador. He has participated in several herpetology projects across the country and has worked in conservation
projects such as the creation of a biological corridor for the Podocarpus National Park, Ecuador.
April 2015 | Volume 8 | Number 1 | e91
Amphib. Reptile Conserv.
140
Official journal website:
amphibian-reptile-conservation.org
Amphibian & Reptiie Conservation
8(1) [Special Section]: 141-142 (e93).
Book Review
The Amphibians and Reptiles of Mindo:
Life in the Cloudforest
^^Howard O. Clark, Jr. and ^''Craig Hassapakis
^Senior Wildlife Ecologist, Garcia and Associates, Clovis, California, USA ^Publisher and Editor, Amphibian & Reptile Conservation; Editor,
ErogLog; lUCN SSC Amphibian Specialist Group and Genome Resources Working Group; Provo, Utah, USA
Key words. Amphibia, Reptilia, Ecuador, conservation, ecotourism, field researeh, eitizen scientist
Citation: Clark HO Jr, Hassapakis C. 2015. Book Review — The Amphibians and Reptiles of Mindo: Life in the Cloudforest. Amphibian & Reptile
Conservation 8{t) [Special Section]; 141-142 (e93).
Copyright: © 2015 Clark and Hassapakis. This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCom-
mercialNoDerivatives 4.0 International License, which permits unrestricted use for non-commercial and education purposes only, in any medium,
provided the original author and the official and authorized publication sources are recognized and properly credited. The official and authorized
publication credit sources, which will be duly enforced, are as follows: official journal title Amphibian & Reptile Conservation] official journal website
<amphibian-reptiie-conservation.org>.
Received: 01 Feburary 2015; Accepted: 14 April 2015; Published: 18 April 2015
Title: The Amphibians and Reptiles of Mindo:
Life in the Cloudforest
Authors: Alejandro Arteaga, Lucas Bustamante,
Juan M. Guayasamin
Copyright: 2013
ISBN: 978-9942-13-496-7
Publisher: Universidad Technologica Indoamerica
Pages: 258; Price: $49.00 (US)
The authors have produced a much needed local field
guide for the Mindo parish, located in northwestern Ec-
uador and set a high standard for future field guides to
follow. The book fills a void with great detail and care. It
begins with the Table of Contents, a Forward, and Pref-
ace. A brief Symbols and Abbreviation page is followed
by the Introduction, which leads into a helpful section
on locating and observing reptiles and amphibians in the
Mindo region. The authors recommend that those inter-
ested in exploring Mindo ’s herpetofauna should do some
homework: understand the habitats and environments
where herpetofauna can be found, know your subject,
keep a low profile, and try not to disturb the sensitive mi-
crohabitats in which these unique species are found. Page
1 1 illustrates some principal identification features of the
amphibians of Mindo (as a diagram figure; p. 11). On the
next few pages are additional figures that show the groin
pattern and color of the Mindo rainfrogs (Pristimantis),
principal scale types of Mindo lizards, dewlap color of
Mindo anoles, and basic terminology for snake scalation.
In total the guide features 20 charts and figures, and 228
superb photographs and art work (a special feature and
highlight of the book). These figures allow the future ex-
plorer of Ecuadoran cloudforests (particularly Mindo) to
prepare for an informed and exciting field trip.
The crux of the book are the accounts. The guide fea-
tures 101 species accounts of Mindo’s unique reptiles
and amphibians, with each account accompanied with,
as mentioned above, outstanding photos and in addition,
a range map. The 228 photos are adequate for identify-
ing the target species and have been photographed with
a white background, eliminating distracting clutter so
the reader can focus on key marks, characteristics, and
colors of each species (see Figure 1 for examples). The
range maps are up-to-date and reflect the most current
research (in total, 4,000 locality records are featured).
Each species account has been peer-reviewed by two or
more experts (71 total reviewers and hundreds of per-
sonal connnunications from experts). The accounts are
divided into several key sections: English and Spanish
common names, Latin name with describing author and
year, recognition information, natural history, distribu-
tion, conservation status, etymology, notes, reviewer
and contributor information, and references. Prior to the
species account sections is the “Plan of the Book” — this
section is a must read in that it explains how species ac-
counts are set up and discusses the rational of account
structure. Additionally, pages 27-29 discusses the Mindo
parish; why the area is worthy of continual conservation,
and describes the unique characteristics of the region that
is home to more than 100 species of reptiles and amphib-
ians in an area smaller than the state of Nevada.
Correspondence. Email: ^hclark® garciaandassociates.com-, ‘^arc.publisher® gmail.com (Corresponding author).
April 2015 | Volume 8 | Number 1
Amphib. Reptile Conserv.
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Clark and Hassapakis
After the species accounts the book presents a de-
scription of a new species of Pristimantis found in Mindo
(Arteaga et al. 2013). This account illustrates that new
species are continually being discovered and regions like
Mindo may harbor other new species.
Following the new species description is the Glos-
sary, Reference section (the book lists 1,935 references
throughout), and the final section is “about the authors,”
listing the scientific illustrators and geographers (Rita
Hidalgo, Silvia Cevallos, and Belen Bans).
Overall, the field guide of Mindo is an outstanding
contribution to the ever-growing field of herpetology,
will help conservation efforts, encourage ecotourism
and nature observation, be a high standard for other field
guides to follow, among other positive allied outcomes,
while linking conservation efforts through its publication.
The guide is pleasing to read and should inspire others to
write and publish regional guides in species rich areas
of the Americas, and, as mentioned several times al-
ready, sets a high standard for others to follow. The book
emphasizes a warning that species extinction is real, is
primarily a result of habitat loss, and areas like Mindo
are not necessarily safe. The future is unknown and with
the advent of climate change, disease, encroachment, as
well as many other detrimental factors not mentioned,
we may be witnessing the last sanctuaries for these one-
of-a-kind species. We need to do our part to spread the
word and conserve what’s left. The publication of a field
guide such as this is very important in bringing attention
to the great variety of unique species and lending impe-
tus to conservation efforts. Field guides like this one are
also synergistic in bringing about increased conservation
efforts and making a positive impact to curb the unprec-
edented rate of habitat loss. We recommend that you sup-
port the conservation of Mindo by purchasing the book,
learning about Mindo’s amphibians and reptiles, and
joining in the conservation efforts of the area (or other
similar areas throughout the world) through ecotourism,
conservation research (e.g., citizen scientists), and other
avenues of endeavors and conservation activism via your
individual expertise and enthusiasm to conserve all life
on earth, including our own species. No matter who we
are (average or exceptional, and all other categories as
well) we all can make a positive difference in protecting
and conserving earth’s unique and precious life systems
and diverse biological life (see also Conrad and Hilchey
Fig. 1. Book cover of The Amphibians and Reptiles of Mindo:
Life in the Cloudforest. Photo by Howard O. Clark, Jr.
2011; Johnson et al. 2014), for which amphibians and
reptiles form an exciting component.
Literature Cited
Arteaga A, Yanez-Munoz M, Guayasamin JM. 2013. A
new frog of the Pristimantis lacrimosus group (An-
ura: Craugastoridae) from the montane forests of
northwestern Ecuador. Serie de Publicaciones Cienti-
ficas 1: 198-210.
Conrad CC, Hilchey KG. 2011. A review of citizen sci-
ence and community-based environmental monitor-
ing: issues and opportunities. Environmental Monitor-
ing and Assessment 176(1-4): 273-291. doi: 10.1007/
S10661-010-1582-5
Johnson MF, Hannah C, Acton L, Popovici R, Karanth
KK, Weinthal E. 2014. Network environmentalism:
Citizen scientists as agents for environmental advo-
cacy. Global Environmental Change 29: 235-245.
http://dx.doi.Org/10.1016/j.gloenvcha.2014.10.006
Howard O. Clark, Jr. is a Certified Wildlife Biologist® with 20 years of professional wildlife experienee. He
focuses his time on the fauna and ecosystems of Northern, Central, and Southern California, and the Mojave
Desert. He regularly works with the San Joaquin Kit Fox, Giant Kangaroo Rat, and the Mohave Ground Squirrel.
He currently volunteers as the Layout Editor for ']o\xma[ Amphibian & Reptile Conservation.
Craig Hassapakis is the publisher and editor of the journal Amphibian & Reptile Conservation (amphibian-
reptile-conservation.org); be is also an editor of FrogLog (www.amphibians.org/froglog/) and is a member of tbe
lUCN SSC Amphibian Specialist Group (ASG) and volunteer coordinator for the Genome Resources Working
Group (ASG/GRWG) in that same organization.
April 2015 | Volume 8 | Number 1
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April 2015 | Volume 8 | Number 1
Amphib. Reptile Conserv.
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e92
Official journal website:
amphibian-reptile-conservation.org
Amphibian & Reptiie Conservation
8(2) [Special Section]: 143-162 (e92).
Amphibians and reptiles of an agroforestry system in the
Colombian Caribbean
^Oscar Angarita-M., ^Andres Camilo Montes-Correa, and ^Juan Manuel Renjifo
Grupo de Investigacion en Manejo y Conservacion de Fauna, Flora y Ecosistemas Estrategicos Neotropicales (MIKU), Universidad del Magdalena,
COLOMBIA
Abstract. — Land-use change is a factor that may alter the assembly of herpetofaunal communities.
To determine the effects of land use change, we characterized the herpetofaunal community of
“La Gloria Project” in Magdalena, Colombia. Agroforestry crops (Red Gum, Pink Trumpet Tree,
Beechwood, and Teak), native forest, wetlands, and built-up zones composing the site. From March
to October 2012, we performed eleven field trips, of ten days (eight hours each) for a total sampling
effort of 880 hours per observer. We implemented visual encounter surveys and pitfall traps for
herpetofauna detection. We recorded 23 amphibian (3,555 individuals) and 37 reptile species (1,088
individuals); the highest diversity for both amphibians and reptiles were found in native forest.
Comparing disturbed areas. Teak agroforest presented the highest diversity for both taxa relative
to non-natural environments, by factors such as big leaf size, generating conditions to sustenance
of some species. However, we demonstrated that short-term differences between natural and non-
natural habitats are significant, since there has not been enough time for generalist species to
displace the susceptible species and occupy their niches in all vegetation coverages in the study
area.
Key words. Agroforest, Caribbean lowlands, habitat fragmentation, herpetofaunal concnnunities, tropical dry forest,
lower Magdalena River
Resumen. — El cambio de usos del suelo es un factor que puede afectar el ensamblaje de
las comunidades de herpetofauna. Para determinar los efectos del cambio del uso de suelo,
caracterizamos la comunidad de herpetofauna del “Proyecto La Gloria” en el departamento del
Magdalena, Colombia. Cultivos agroforestales (eucalipto rojo, roble rosado, melina y teca), bosques
natives, humedales y zonas con construccion constituyen el area de estudio. De marzo a octubre de
2012, desarrollamos once salidas de campo de diez di'as (cada uno de echo horas) per un esfuerzo
total de muestreo total de 880 horas x observador. Utilizamos busqueda libre per encuentro casual
y trampas de caida para la deteccion de herpetofauna. Registramos 23 anfibios (3,555 individuos) y
37 reptiles (1,088 individuos); La mayor diversidad tanto para anfibios como reptiles la encontramos
en los bosques natives. Comparando las areas intervenidas, el agrobosque de teca presento la
mayor diversidad de ambos taxones con respecto a los otros ambientes no naturales, por factores
como el gran tamaho de sus hojas, que generan condiciones para el sostenimiento de algunas
especies. Empero, se demuestra que a corto plazo, las diferencias entre los habitats naturales y no
naturales son significativas, pues no ha pasado suficiente tiempo para que las especies generalistas
desplacen a la especies sensibles y ocupen sus nichos.
Palabras clave. Agrobosques, bajo rio Magdalena, bosque seco tropical, comunidades de herpetofauna, fragmentacion
de habitat, tierras bajas del Caribe
Citation: Angarita-M O, Montes-Correa AC, Renjifo JM. 2015. Amphibians and reptiles of an agroforestry system in the Colombian Caribbean. Am-
phibian & Reptiie Conservation 8{2) [Special Section]: 143-162 (e92).
Copyright: © 201 5 Angarita-M et al. This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-
NoDerivatives 4.0 International License, which permits unrestricted use for non-commercial and education purposes only, in any medium, provided
the original author and the official and authorized publication sources are recognized and properly credited. The official and authorized publication
credit sources, which will be duly enforced, are as follows: official journal title Amphibian & Reptiie Conservation] official journal website <amphibian-
reptiie-conservation. org> .
Received: 22 January 2015; Accepted: 09 April 2015; Published: 15 April 2015
Correspondence. Email: ^oscarangaritabio@ gmail.conr, ^andresc.montes® gmail.com (Corresponding author);
^juanmanuel. renjifo @ gmail. com
April 2015 | Volume 8 | Number 1
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Angarita-M et al.
Introduction
Colombia ranks second in taxonomic diversity of am-
phibians (785 species) and third in reptiles (593 species)
(Acosta-Galvis 2014; Andrade-C. 2011). In the Caribbe-
an lowlands 167 reptiles species and 55 amphibians are
recorded (Romero-Martmez and Lynch 2012; Carvajal-
Cogollo et al 2012). The low diversity of amphibians in
the Colombian Caribbean is due to drier conditions of
the region, however, the small number of species have
morphological, physiological, and behavioral adapta-
tions to tolerate drought (Cuentas et al. 2002). Existing
surveys include checklists, inventories, and diversity of
amphibians and reptiles for the entire region (Dugand
1975; Carvajal-Cogollo et al. 2012; Romero-Martmez
and Lynch 2012); as well as the states of Cordoba (Ren-
jifo and Lundberg 1999; Carvajal-Cogollo et al. 2007;
Carvajal-Cogollo and Urbina-Cardona 2008; Romero-
Martmez et al. 2008; Romero-Martmez and Lynch 2010),
Sucre (Galvan-Guevara and de la Ossa- Velasquez 2009;
Acosta-Galvis 2012b), Bolivar and Atlantico (Cuentas et
al. 2002), Cesar (Rueda-Almonacid et al 2008a; b; Me-
dina-Rangel 2011; Medina-Rangel et al. 2011), La Gua-
jira (Galvis et al. 2011; Blanco-Torres et al. 2013), and
Magdalena (Ruthven 1922; Duenez-Gomez et al. 2004;
Rueda-Solano and Castellanos-Barliza 2010; Montes-
Correa et al. 2015). Many studies were performed in nat-
ural areas with wetlands or native forests, with different
levels of anthropogenic intervention. Nonetheless, the
information on the herpetofauna of dry spots is scarce,
and most of the available literature are species descrip-
tions, taxonomic reviews of specific groups, or national
lists (Acosta-Galvis, 2012a).
Deforestation and changes in land-use modify the
assembly of amphibian and reptile communities (Cas-
tro and Kattan 1991; Garden et al, 2007). The physical
transformation of natural environments can cause drastic
changes in humidity and temperature, having significant
effects in these organisms (Herrera et al. 2004). How-
ever, dryland amphibians have several adaptations to sur-
vive the lack of water, as the changes in activity patterns
and development of wide ranges of dehydration (Thor-
son 1995; Cuentas et al. 2002).
Moreover, reptiles are more resistant to disturbance as
their skin is covered by keratinized scales. Anmiotic eggs
make reptiles more tolerant to dehydration and sunstroke
(Vargas-Salinas and Bolanos 1999). Even so, the canopy
cover, leaf litter cover, and understory density are impor-
tant factors for the establishment and distribution of both
taxa, since it can determine the movement patterns of
these ectothermic animals (Urbina-Cardona et al. 2006).
Our goal was to determine the diversity of herpeto-
fauna in “La Gloria Project” (Sabanas de San Angel,
Magdalena, Colombia), and assess the characteristics
and variations of herpetofaunal communities among the
various vegetation coverages (Agroforestry crops — Red
Gum, Pink Trumpet Tree, Beechwood, and Teak — native
forest, wetlands, and built-up zones [any area inhabited
by humans] composing the area).
Materials and Methods
Study site: “La Gloria project” is part of “Reforestadora
de la Costa (REEOCOSTA S.A.S.)” organization, within
the jurisdiction of the municipality of Sabanas de San
Angel, Magdalena department, 30 km from the county
seat (10°10’29.2”N; 74°19’38.052”W) (Pig. 1). The
study area includes 7,288 hectares, and corresponds to
“zonobioma tropical altemohigrico” (tropical dry forest)
proposed by Hemandez-Camacho and Sanchez (1992).
This locality has a unimodal biseasonal climate with an
average annual rainfall of 1,157 mm (Rangel-Ch. and
Carvajal-Cogollo 2012). The oldest agroforest is about
about 20 years old. Timber is grown in the middle ex-
tension of the La Gloria project. The main crop is Teak
(Tectona grandis) (21% of the total extent of study area),
followed by Red Gum {Eucalyptus tereticornis) (18%).
Also grown to a lesser extent is. Pink Trumpet Tree {Ta-
bebuia rosea) (7%) and Beechwood (Gmelina sp.) (2%),
Caribbean Sea
ftVROWA
SAMTA M'AkI
ik’lDTalj*/
WA.PA. PROYECTO LA GLORIA
mi Red Gum
□ Pink Trumpet T
mi Beechwood
] t Teak
Native forest
mi Wetland
Built-up zones
Other Crops
CZJi Teak regrowth
L_l Pasture
ESC: 1:SS.DD0
Leyenda
Study area
Nationals Parks
1 : 1 . 000,000
Fig. 1. Map of La Gloria Project (taken and modified from Re-
focosta 2012). Map developed by HD Granda-Rodriguez.
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Herpetofauna of an agroforestry system in the Colombian Caribbean
while the remaining 2% consists of other crops. In addi-
tion to agroforests, there is an area of regrowth of Teak
(5%), pasture (34%), native forest (10%), and wetlands
(1%) (Refocosta 2012). There are also small and scat-
tered built-up zones within “La Gloria Project.” Surveys
for this study were carried out in areas with agroforest,
native forests, wetlands, and urbanized sites.
Fieldwork: from March to October of 2012, we made 11
field trips, each one lasting ten days. We used Visual En-
counter Surveys (VES) (Crump and Scott 1994). Daily, a
single person did random walks for eight hours (09:00-
12:00, 14:00-17:00, and 19:00-22:00 h), for a total sam-
pling effort of 880 hours x observer. In addition, we cap-
tured cryptic species with terrestrial, semifosorial, and
fossorial habits with pitfall traps (Vogt and Mine 1982),
eight trap systems per habitat during each survey (56 in
total). These traps system consist of two 3.78 liters buck-
ets, and a two m interception net between them. Traps
remained open for ten days.
We used a 10% chlorobutanol solution to euthanize all
amphibians captured and intrathoracic lidocaine injec-
tions for euthanizing reptiles. No turtles or crocodilians
were sacrificed for this study. All vouchered specimens
were deposited in the Centro de Colecciones Biologicas
de la Universidad del Magdalena (CBUMAG:REP and
CBUMAG:ANF acronym). The scientific nomenclature
used in this contribution is that accepted by Uetz et al.
(2014) and Frost (2014).
Data analysis: Relative abundance was calculated as the
number of individuals in each sample relative to capture
effort, expressed in individuals/hours x observer (RA=
Ind/h X obs.) (Lips 1999). Species were qualified accord-
ing to their relative abundance in “very rare” (VR) if it
was observed between 0.1-0.24 individuals per hour x
observer; “rare” (R) if it was observed between 0.25-
0.49; “common” (C) if it was observed between 0.50-
0.74; “abundant” if it was observed between 0.75-0.99;
and “very abundant” if it was observed between 1.0 or
more (Rueda-Solano and Castellanos-Barliza 2010). Us-
ing PRIMER 6 (v 6.1.11) (Clarke and Corley 2001) we
calculated Margalef Richness Index (d), Pielou Unifor-
mity Index (J’), Shannon- Wiener Diversity Index (H’),
and Simpson Dominance Index (X) for each vegetal
coverage. We built a Bray-Curtis Similarity Matrix of
non-transformed amphibian and reptile abundance data,
to generate a nonparametric one-way similarity analysis
(ANOSIM) (999 permutations), in order to refute a null
hypothesis when there were no significant differences be-
tween diversity of amphibians and reptiles among sites.
We made dendrograms with the same Bray-Curtis Ma-
trix, to evaluate the similarity among vegetal coverages
within the study area; likewise, the similarity between La
Gloria project and other localities with published inven-
tories of amphibians and reptiles in the Colombian Carib-
bean. It should be noted that if the similarity was greater
than 50%, it was considered a homogenous cluster. We
used the software Estimates (v 9.1.0) (Coldwell 2013) to
create a species accumulation curve from non-parametric
qualitative estimators Chao 2, Bootstraps, Jacknife 1, and
Jacknife 2 (randonfized 999 times for each case) to quan-
tify the representativeness of the sample. We also calcu-
lated the unique and duplicates species.
Results and Discussion
Representativeness of survey: Bootstraps, Chao 2, Jack-
nife 1, and Jacknife 2 estimators show that amphibian
survey had representativeness among 24.83% to 28.95%.
The Chao 2 curve was only one who got stabilization.
The unique and duplicated species were not reduced
during the survey (Fig. 2a). Furthermore, the reptile sur-
veys had more representativeness, since the estimators
reached among 39.79% to 45.94%. The Chao 2 and Jack-
nife 2 curve obtained asymptote. In this case, unique and
duplicates species neither decreased (Fig. 2b). Jacknife 1
and Jacknife 2 estimators have higher values, suggesting
that surveys had a low representativeness in both taxa
(Carvajal-Cogollo and Urbina-Cardona 2008). Boot-
straps estimator obtained close values with observed
species. Taking this as a reliable algorithm to estimate
total richness, amphibian and reptile surveys reached a
representativeness of 24.83% and 39.79% respectively.
A comparison of survey methods used (observational
surveys [VES] and trapping) results in a greater number
of species and abundance being obtained through VES.
(Fig. 3a, b). Using this technique, we detected 92.31%
0 20 40 eo so 100 120
#3 Ivfean(nms) ■UmqufisMfian * Duplicates Mean Chao 2 Mean
KJack 1 Mean • Jack 2 Mean -l-BootstrapMfean
Fig. 2. Cumulative curve species of la Gloria project.
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Angarita-M et al.
Table 1. Relative abundance = RA, VA = very abundant, A = abundant, C = common, R = rare, VR= very rare, NA = not avail-
able, and vegetation coverage, RG = Red Gum, PTT = Pink Trumpet Tree, BW= Beechwood, T = Teak, NF = native forest, WL =
wetland, BZ = built-up zones. CBUMAG = Centro de Colecciones Biologicas de la Universidad del Magdalena (ANF =
amphibian; REP = reptile).
TAXA
GT
PTT
BW
T
NF
WL
BZ
RA
Voucher
CLASS AMPHIBIA
CBUMAG:ANF
Order Anura
Family Bufonidae
Rhinella marina (Linnaeus 1758)
X
X
X
X
X
X
R
699
Rhinella humboldti (Gallardo 1965)
X
X
X
X
X
VR
701
Family Ceratophrydae
Ceratophrys calcarata (Boulenger 1890)
Family Hylidae
X
VR
672
Dendropsophus microcephalus (Cope 1886)
X
X
VA
713
Dendropsophus ebraccatus (Cope 1874)
X
VR
00666-67
Hypsiboas pugnax (Schmidt 1857)
X
X
X
VA
00697-8
Hypsiboas crepitans (Wied-Neuwied 1824)
X
VR
30
Scarthyla vigilans (Solano 1971)
X
X
VA
718
Scinax rostratus (Peters 1863)
X
X
VR
00031-32, 49
Scinax “x-signatus” (Spix 1824)
X
X
X
X
X
R
15
Trachycephalus typhonius (Linnaeus 1758)
X
X
VR
696
Phyllomedusa venusta Duellman and Trueb 1967
X
VR
676
Pseudis paradoxa (Linnaeus 1758)
Family Leptodactylidae
X
VR
Leptodactylus fuscus (Schneider 1799)
X
X
X
X
X
R
00703-4
Leptodactylus insularum Barbour 1906
X
X
X
X
R
00693, 695, 700
Leptodactylus poecilochilus (Cope 1862)
X
X
VR
348
Leptodactylus fragilis (Brocchi 1877)
X
VR
Engystomops pustulosus (Cope 1864)
X
X
X
X
X
R
00708,711,716
Pleurodema brachyops (Cope 1869)
X
X
X
X
C
00702, 705
Pseudopaludicola pusilla (Ruthven 1916)
Family Microhylidae
X
X
X
X
C
00709, 717
Elachistocleis panamensis (Dunn, Trapido, and Evans 1948)
X
X
VR
719
Elachistocleis pearsei (Ruthven 1914)
Order Gymnophiona
Family Caecilidae
X
X
X
X
VR
00710, 720
Caecilia subnigricans Dunn 1942
X
VR
634
CLASS REPTILIA
CBUMAG:REP
Order Squamata
Family Sphaerodactylidae
Gonatodes albogularis (Dumeril and Bibron 1836)
X
X
X
X
X
VR
236
Lepidoblepharis sanctaemartae (Ruthven, 1916)
X
X
VR
Family Gekkonidae
Hemidactylus frenatus (Dumeril and Bibron 1836)
Family Phyllodactylidae
X
VR
237
Thecadactylus rapicauda (Houttuyn 1782)
Family Iguanidae
X
X
VR
Iguana iguana (Linnaeus 1758)
Family Dactyloidae
X
VR
Anolis auratus Daudin 1 802
X
VR
231
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Herpetofauna of an agroforestry system in the Colombian Caribbean
Table 1 (Continued). Relative abundance = RA, VA = very abundant, A = abundant, C = common, R = rare, VR = very rare, NA =
not available), and vegetation coverage, RG = Red Gum, PTT = Pink Trumpet Tree, BW = Beecbwood, T = Teak, NF = native for-
est, WL = wetland, BZ = built-up zones. CBUMAG = Centro de Colecciones Biologicas de la Universidad del Magdalena.
TAXA
GT PTT
BW
T
NF
WL BZ
RA
Voucher
Family Corytophanidae
Basiliscus basilicus (Linnaeus 1758)
Family Scincidae
X
VR
Maracaiba zuliae (Miralles, Rivas, Bonillo, Schargel, Barros,
Garcia-Perez, and Barrio- Amoros 2009)
X
X
X
X
VR
235
Family Gymnophthalmidae
Leposoma rugiceps (Cope 1869)
X
X
X
VR
239
Tretioscincus bifasciatus (Dumeril 1851)
Family Teiidae
X
X
X
X
VR
00232-33
Cnemidophorus gaigei Ruthven 1915
X
X
R
Ameiva praesignis (Baird and Girard 1852)
X
X
R
Ameiva bifrontata Cope 1862
Family Anomalepididae
X
R
Liotyphlops albiwstris (Peters 1857)
Family Boidae
X
VR
194
Boa constrictor Linnaeus 1758
X
VR
Epicrates maurus Gray 1849
Family Colubridae
X
VR
234
Chironius spixii (Hallowell 1845)
X
X
VR
120
Tantilla melanocephala (Linnaeus 1758)
X
VR
00208, 210
Leptophis ahaetulla (Linnaeus 1758)
Family Dipsadidae
X
VR
10
Leptodeira annulata (Linnaeus 1758)
X
X
VR
34
Leptodeira septentrionalis (Kennicott 1859)
X
X
X
VR
Lygophis lineatus (Linnaeus 1758)
X
X
VR
Pseudoboa neuwiedii (Dumeril, Bibron, and Dumeril 1 854)
X
X
VR
91
Imantodes cenchoa (Linnaeus 1758)
X
X
VR
16
Thamnodynastes gambotensis Perez-Santos and Moreno 1989
X
X
NA
232
Thamnodynastes paraguanae Bailey and Thomas 2007
X
X
NA
38
Helicops danieli Amaral 1938
X
VR
128
Oxyrhopus petolarius (Linnaeus 1758)
X
VR
238
Xenodon rabdocephalus (Wied 1824)
Family Viperidae
X
VR
00170-71
Crotalus durissus Linnaeus 1758
X
VR
Porthidium lansbergii (Schlegel 1841)
X
VR
74
Bothrops asper (Garman 1883)
Family Elapidae
X
X
VR
165
Micrurus dissoleucus (Cope 1860)
X
VR
Order Testudines
Family Chelidae
Mesoclemmys dahli (Zangerl and Medem 1957)
Family Emydidae
X
VR
Trachemys callirostris (Gray 1855)
X
VR
Eamily Testudinidae
Chelonoidis carbonarius (Spix 1824)
Order Crocodylia
Eamily Alligatoridae
X
X
VR
Caiman crocodilus (Linnaeus 1758)
X
A
Amphib. Reptile Conserv. 148 April 2015 | Volume 8 | Number 1 | e92
Angarita-M et al.
of amphibian and 68.48% of reptile individuals, respec-
tively. With VES, we recorded 21 amphibian species
and 35 reptile species. With this method we recorded 25
exclusive species (10 amphibians and 15 reptiles), that
are strictly arboreal or aquatic. Conversely, we captured
7.69% and 31.52% of amphibian and reptile individuals
respectively, using pitfall traps. This method recorded 12
amphibian species and 19 reptile species. We only found
two fossorial species {Elachistocleis pearsei and Micru-
rus dissoleucus) with pitfall traps.
Amphibians: A total of 3,555 individuals, corresponding
to two orders, six families, and 23 species (Table 1), were
recorded. Anurans found represented five families and 22
species (37% of the total herpetofauna of the area) (Fig
4); a single caecilian specie was encountered (Fig. 4).
Forty-two percent (41.8%) of lowland amphibian species
occurring in the Colombian Caribbean were observed at
Fa Gloria Project. The absence of expected species is due
to a lack of specialized capture methods. For example,
Typhlonectes natans is rarely observed due to its cryp-
tic aquatic habits despite being distributed throughout
the Caribbean region of the upper Magdalena-Cauca
River (Tapley and Acosta-Galvis 2010). However, in this
study we report the first record of the Clown Treefrog
(Dendropsophus ebraccatus) in the lower Magdalena
River, for which the nearest known distribution is in Rio
Manso, Cordoba (Cochran and Coin 1970). In this con-
tribution, we prefer to name Scinax "‘x-signatus’' instead
Scinax ‘"rubef' (as was known previously Renjifo and
Fundberg 1999; Cuentas et al. 2002). This is due to unre-
solved controversy regarding its taxonomy and biogeog-
raphy (Barrio- Amoros 2004; Acosta-Galvis et al. 2006;
Barrio-Amoros et al. 2011; Acosta-Galvis et al. 2012a).
Following Rivero’s (1969) criteria, the absence of dark
dorsolateral lines and head equally long as wide place
the collected specimens within the x-signatus and ruber
groups.
Reptiles: We recorded 1,088 specimens corresponding to
three orders, 19 fanfilies, and 37 species (Table 1). The
most diverse order was Squamata with 15 fanfilies and
32 species, the suborder Facertilia was the richest with
nine families and 13 species, 20% of the total herpeto-
fauna of the area. The suborder Serpentes represented
six fanfilies and 20 species (34%). We observed three
families and three species of turtles (5%) and recorded
one crocodilian species (2%) (Fig. 3). Fa Gloria Project
harbors 21.8% of lowlands reptile species of the Colom-
bian Caribbean. We found three endenfic species from
Colombia, Helicops danieli, Thamnodynastes gamboten-
sis, and M. dahli, the latter with restricted distribution
in the Colombian Caribbean (Rossman 2002; Bailey and
Thomas, 2007; Carvajal-Cogollo et al. 2012; Forero-Me-
dina et al. 2013). The presence of M. dahli in the study
area was unexpected, as species distribution models by
Forero-Medina et al. (2012) propose a low probability
a)Amphibians
100
b) Reptiles
so
70
Richness Abundance (%) Exclusive species
■ PitfaUTiape iVES
Fig. 3. Comparisons between the methods used for herpeto-
fauna recording and capturing.
Squainata: Seipentes
34 %
Testudines Crocodylia
5 % * 2 %
Squamata: Lacertlia
20 %
Fig. 4. Herpetofauna composition percentage in la Gloria Proj-
ect.
of occurrence within this region. However, this area has
many first-order streams with abundant riverine vegeta-
tion, throughout native forests and agroforests, habitat
characteristics of this species (Forero-Medina et al. 2011;
Montes-Correa et al. 2014).
In addition, we report the first record of Maracaiba
zuliae in the lower Magdalena River, an expansion of its
currently known distribution. This species was recently
reported in Colombia in Reserva Forestal Protectora
Montes de Oca, Fa Guajira state (Galvis et al. 2011).
Several records by Ruthven (1922) in the Barbacoas Riv-
er, the Arenas Stream, and Fas Pavas must correspond
with this recently described species. Fikewise, we report
the first record of Thamnodynastes paraguanae in the re-
gion. In Colombia, this snake is only known from Fa Gu-
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Herpetofauna of an agroforestry system in the Colombian Caribbean
a) Richness
30
RG PTT BW T NF WL BZ
■ Amphibiaiis ■ Reptiles
Fig. 5. Richness (a) and abundance (b) of amphibians and rep-
tiles between habitats (RG = Red Gum, PTT = Pink Trumpet
Tree, BW = Beechwood, T = Teak, NF = native forest, WL =
wetland; BZ = built-up zones).
ajira: Uribia, Riohacha, and Reserva Forestal Protectora
Montes de Oca (Bailey and Thomas 2007; Galvis et al.
2011). We must clarify that while the fieldwork was de-
veloped, snakes of Thamnodynastes genus were treated
as one species, and they are not included in this analysis
because their relative abundance is not available.
Richness and abundance patterns: in La Gloria Project,
the native forest was the habitat that hosted the greatest
number of species (Fig. 5a), 18 amphibians and 26 rep-
tiles. The wetlands were the second habitat in amphib-
ian composition, while the Teak agroforest was second
in number of reptiles. Remaining habitats had less or
equal to 10 species, both for amphibians and reptiles. We
verified the greatest abundance in native forest (Fig. 5b),
as 65.63% of amphibians and 48.35% of reptiles were
detected in this habitat. All non-natural habitats scored
an abundance below 10%. Some studies show that abun-
dance patterns of natural and non-natural environments
are similar (Gardner et al. 2007; Carvajal-Cogollo and
Urbina-Cardona et al. 2008). Over time, composition
and abundance tend to homogenize by dominance of the
generalist species that displace more sensitive species
for their lower habitat requirements and increased toler-
ance to disturbance (Offerman et al, 1995; Laurance et al.
2002). Surely, La Gloria Project does not present homog-
enization because agroforests are very recent. Regarding
the qualitative relative abundance in amphibians, we ob-
served three very abundant species, two common, five
rare, and 13 very rare. Dendropsophus microcephalus,
Scarthyla vigilans, and Hypsiboas pugnax were the most
abundant amphibians, while Caecilia subnigricans is
represented by a single individual. Moreover, in reptiles
we observed one abundant species, three rare, 31 very
rare, and two not available. The most abundant species
of reptiles were Caiman crocodilus, Ameiva bifrontata,
mdAmeiva praesignis. Furthermore, Mesoclemmys dah-
li and Micrurus dissoleucus were observed for a single
individual. Similarly, other studies of tropical dry forest
herpetofauna, found over half species had low relative
abundance (Rueda-Solano and Castellanos-Barliza 2010;
Pedroza-Banda and Angarita-Sierra 2011). In addition,
snakes present a lower detection, possibly due to their
cryptic habits or low abundance. Leptodeira annulata
and Leptodeira septentrionalis were the most common
snakes throughout the study area, supporting Scott and
Seigel (1992) and Dodd (1993) hypotheses, where small
sized snakes are more tolerant to disturbance, therefore,
possibly more abundant. As to the connnunity attributes
(Table 2), native forest had the highest Margalef Rich-
ness and Shannon-Wiener Diversity for amphibians
and reptiles and Beechwood agroforest had the greatest
Pielou Uniformity Value. For these three attributes, built-
up areas showed the lower values, however, this habi-
tat had dominance for the highest values. In this study,
the higher value of Margalef Richness, Shannon- Wiener
Diversity, and Pielou Uniformity created higher values
obtained for the coverage of floristic and structural com-
plexity. A similar pattern was observed in Zapatosa re-
gion by Medina-Rangel (2011).
Habitat comparisons and herpetofaunal autoecology:
ANOSIM determined there are global composition and
abundance differences between seven evaluated habitats
(p- value = 0.502). However, there are specific differ-
ences between Red Gum agroforest and Pink Trumpet
Tree agroforest (p-value = 0.006), Red Gum and Beech-
wood (p-value = 0.038), Red Gum and Teak (p-value =
0.161), Pink Trumpet Tree and Beechwood (p- value =
0.068), Pink Trumpet Tree and Teak (p-value = 0.012),
and Beechwood and Teak (p-value = 0.357). These simi-
larities among agroforests are due to sharing among pio-
neer and generalist species that are able to tolerate condi-
tions imposed by the new environment (Luja et al. 2008),
e.g.. Nest-building Frogs {Leptodactylus) (Heyer 1969).
Some of these can be considered as connnon colonizers
(see also, Duenez-Gomez et al. 2004).
In La Gloria Project, the herpetofauna composition
was quite heterogeneous, thus, all clusters were below
50% similarity (Fig 6). The more similar habitats were
the Teak and Pink Trumpet Tree (48.5% similarity).
Likewise, native forests and wetlands have a cluster
(42.6%) and Beechwood and Red Gum agroforest an-
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Angarita-M et al.
BW
RG
T
prr
WL
NF
BZ
0 20 40 60 80 100
Similarity
Fig. 6. Bray-Curtis similarity dendrogram between habitats in la Gloria Project (RG = Red Gum, PTT = Pink Trumpet Tree, BW =
Beechwood, T = Teak, NF = native forest, WL = wetland; BZ = built-up zones).
Universidad del Magdalea
Besotes
Neguanje
Cordoba wetlands
La Gloria
Montes de Marfa and La Caimanera
Zapatosa
Atlantico and Bolivar
El Botillero
Montes de oca
Rancherfa
Urra
Coraza
Murrucucu lowlands
1 ^ ^ ^ 1
20 40 60 80 100
Similarity
Fig. 7. Similarity of amphibian richness between La Gloria project and others inventories in Caribbean lowlands. Humedales
del Cordoba (Romero-Martrnez and Lynch 2010); Montes de Marfa and Cienaga la Caimanera (Acosta-Galvis 2012b); El Botil-
lero (Duenez-Gomez et al. 2004); Cienaga del Zapatosa (Medina-Rangel et al. 2011); Atlantico and north Bolivar (Cuentas et al.
2002); Montes de Oca (Galvis et al. 2011); Rancherfa (Blanco-Torres et al. 2013); Urra (Renjifo and Lundberg 1999); Los Besotes
(Rueda-Almonacid et al. 2011a); Serranfa de Coraza (Galvan-Guevara and de la Ossa- Velasquez 2009); Universidad del Magdalena
(Montes-Correa et al. 2015); Ensenada Neguanje (Rueda-Solano and Castellanos-Barliza 2010); Cerro de Murrucucu lowlands
(Romero-Martfnez et al. 2008).
other (36.8%). The more dissimilar habitat is the built-up
zone with 3.1% similarity with respect to other habitats.
The species with greater frequency of occurrence was
Rhinella marina, which was present in six of the seven
evaluated habitats. This species has ecological plastic-
ity and is able to tolerate highly degraded environments,
including benefiting from human activities (Zug and
Zug 1979). On the other hand, we found 28 exclusive
species from a single cover. For example, Pseudis para-
doxa. Caiman crocodilus, and Trachemys callirostris are
strictly aquatic species and only found in wetlands. The
exclusivity of Hemidactylus frenatus is due to its strong
synanthropy (Caicedo-Portilla and Dulcey-Cala 2011).
Phyllomedusa venusta and Trachycephalus typhonius
were exclusive of native forests, since these organ-
isms have behavioral adaptations to tolerate prolonged
drought in these habitats (Cuentas et al. 2002).
The richness and abundance of amphibians in La Glo-
ria project was higher in native forests and their nearby
wetlands. Moreover, in the Red Gum agroforest, amphib-
ian richness and abundance was lower due to the sparse
canopy of this tree which allows more sunlight to reach
the forest floor, similar to what Gardner et al. (2007)
reported for Brazil. In Indonesia, Wanger et al. (2009)
Amphib. Reptile Conserv.
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Herpetofauna of an agroforestry system in the Colombian Caribbean
Zapatosa
Humedales del Cordoba
El Botillero
La Gloria
Urra
Coraza
Universidad del Magdalena
Neguanje
Montes de Oca
Rancheria
Besotes
50
60
70
80
90
Similarity
100
Fig. 8. Similarity of reptile richness between La Gloria project and others inventories in Caribbean lowlands. Humedales del
Cordoba (Carvajal-Cogollo et al. 2); El Botillero (Duenez-Gomez et al. 2004); Cienaga del Zapatosa (Medina-Rangel et al. 2011);
Montes de Oca (Galvis et al. 2011); Rancheria (Blanco-Torres et al. 2013); Urra (Renjifo and Lundberg 1999); Los Besotes (Rue-
da- Almonacid et al. 2011b); Serrama de Coraza (Galvan-Guevara and de la Ossa- Velasquez 2009); Universidad del Magdalena
(Montes-Correa et al. 2015); Ensenada Neguanje (Rueda-Solano and Castellanos-Barliza 2010).
found that amphibians are more abundant in native rain-
forests than in Cacao Tree agroforest. In Gorgona Island,
amphibians were more abundant in little disturbed rain-
forests than in palm cultivation (Urbina-Cardona and
Londono-Murcia 2003). On the other hand, in mountain
rainforest, amphibian composition and abundance were
higher in open areas that agroforest and native forests
(Hoyos-Hoyos et al. 2012).
Canopy coverage may not be as important to some
reptiles. Wanger et al. (2009) found that reptile richness
and abundance was similar in Cacao Tree agroforest, na-
tive rainforests and open areas, and even these showed
greater richness in open areas than in native rainforests.
In Gorgona Island, reptile richness was higher in second-
ary forests; nevertheless, were more abundant in dis-
turbed areas than in primary forests (Urbina-Cardona and
Londono 2003). In La Gloria project, the reptile richness
was higher in native forests, although we recorded sev-
eral species in agroforests, mainly in Teak; this because
large leaves of this tree generate heavy shade and leaf-lit-
ter layers able to generate favorable microclimatic condi-
tions for herpetofaunal establishment. In other agroforest
reptile composition and abundance was low due to thin
canopy cover and insufficient leaf-litter depth. In the case
of Red Gum agroforest, the leaf-litter layer is very poor,
as this tree is perennial. Changes of leaf-litter dynamics
can alter amphibian and reptile assembly (Whitfield et
al. 2014).
In La Gloria project, there are typical species of forest
formations, but not necessarily exclusive of native forest.
For example, Lepidoblepharis sanctaemartae occurred
in native forest and Teak agroforest, being slightly more
abundant in the native forest; L. sanctaemartae, as other
small leaf-litter geckos, requires a leaf-litter layer con-
taining humidity and little light penetration through the
canopy, because of their passive thermoregulatory strat-
egy (Vitt et al. 2005). Because of this aspect, L. sanc-
taemartae was not present in Red Gum agroforest. This
species is a good model of Garden et al. (2007) hypoth-
eses, since a dense canopy and a humid leaf-litter layer
are more important for this species persistence than for-
est vegetation composition. Therefore, L. sanctaemartae
is abundant both in preserved native forests as agroforest
with sufficient coverage canopy and leaf-litter humidity
(Montes-Correa pers. obs.).
The tortoise Chelonoidis carbonarius was present al-
most exclusively in native forest, where there is avail-
able fruit, which makes up much of their diet (Rueda-
Almonacid et al. 2007). A single individual was recorded
in Pink Trumpet Tree agroforest, feeding on flowers of
this tree in breeding season, which are also an important
part of their diet (Moskovits and Bjomdal 1990). We did
not find this species in other agroforests since the tim-
ber cultivation does not offer alimentary resources. The
slider turtle Trachemys callirostris was more abundant in
wetlands with open areas on its banks, as these offered
sites for nesting (Moll and Legler 1971).
The Spectacled caiman (C. crocodilus) was very
abundant, being present in all wetlands in the zone. The
low metabolic rate and generalist feeding habits allow
them to maintain populations in areas with small and dis-
perse wetlands (Castro-Herrera et al. 2013). Likewise, it
is possible that the extermination of Crocodylus acutus
in the lower Magdalena River has favored the increasing
April 2015 I Volume 8 | Number 1
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Angarita-M et al.
Table 2. Attributes of amphibians and reptiles communities in the habitats of La Gloria project (d = Margalef richness, J’ = Pielou
Uniformity, H’ = Shannon- Wiener Diversity, X = Simpson Dominance).
Red Gum
Pink Trumpet Tree
Beechwood
Teak
Native Forest
Wetiands
Buiit-up Zone
Amphibians
d
2.03
1.11
0.92
1.33
2.19
2.18
0.8
J'
0.85
0.81
0.93
0.77
0.68
0.55
0.75
H'
0.85
0.57
0.56
0.65
0.85
0.67
0.36
X
0.17
0.31
0.3
0.27
0.2
0.32
0.51
Reptiles
d
0.96
1.55
1.12
3.3
3.83
0.69
0.22
J'
0.82
0.92
0.92
0.84
0.7
0.23
0.31
H'
0.39
0.72
0.44
1.01
0.98
0.16
0.09
X
0.47
0.21
0.39
0.12
0.17
0.85
0.89
populations of C. crocodilus. A similar situation occurred
in Venezuelan Llanos with Crocodylus intermedius ex-
termination (Medem 1981).
Compared to other inventory studies in the Colom-
bian Caribbean lowlands. La Gloria project presented
similarity in richness of amphibians with other inven-
tory studies in areas with abundant wetlands (Fig. 6); it
showed the highest similarity with the Humedales del
Cordoba (Romero and Lynch 2010) (85.7% similarity).
Although they agreed in many lowlands species, forest
formations are scarce in Cordoba Wetlands, thus, in La
Gloria project forest species such as Phyllomedusa ve-
nusta were present, while in Cordoba Wetlands it was
not reported. There is another great cluster with the lo-
calities of La Guajira. Studies made in Urra (Renjifo
and Lundberg, 1999), Coraza (Galvan-Guevara and de
la Ossa- Velasquez 2009), and Murrucucu (Romero et al.
2008) suggest the area of influence of the Sinu River has
many common elements with the Cordillera Occidental,
biogeographic Choco, and Central America, (v. gr. Co-
lostethus pratti, Strabomantis bufoniformis, Bolitoglossa
biseriata, and Oscaecilia polizona). Clustering between
Neguanje (Rueda-Solano and Castellanos-Barliza 2010),
and Universidad del Magdalena (Montes-Correa et al.
2015) and Besotes (Rueda-Almonacid et al. 2008a) is
due to the typical elements of tropical dry forest and the
Sierra Nevada de Santa Marta (as Colostethus ruthveni,
Cryptobatrachus boulengeri, and Allobates sp.).
In reptiles, La Gloria project is very similar to other
areas of lowlands with wetlands, presenting the most
similarity between Humedales del Cordoba (Carvajal-
Cogollo et al. 2007) and Cienaga del Zapatosa (Medi-
na-Rangel et al. 2011) (69.8% similarity) (Fig. 7). This
evident clustering of the lowlands is very similar to the
localities in La Guajira but differs from typical elements
from northeastern Caribbean, as Gonatodes vittatus, Ba-
chia talpa, and Thamnodynastes paraguanae. The west-
ern regions are very dissimilar to La Gloria project by
having typical elements of biogeographic Choco, as Che-
lydra acutirostris and Anolis vittigerus (Medem 1977;
Castro-Herrera and Vargas-Salinas 2008).
Conclusions
This study shows that connnunities of amphibians and
reptiles are affected by structural changes in forests,
since cultivated timber does not provide the necessary
microhabitats to sustain many elements of herpetofauna
species. The introduction of agroforests results in al-
terations of the spatial distribution of species, restricting
them to small remnants of native forest.
A greater problem of studies of amphibians and rep-
tiles in the Colombian Caribbean is that the predominant
information is unpublished literature and the method-
ologies unclear (Blanco-Torres et al. 2013). This study
contributes to the state of knowledge of amphibian and
reptile richness in the lower Magdalena River, provid-
ing three new records for the region and establishes a list
from a standardized inventory.
Acknowledgments. — We thank the company of Re-
focosta S.A.S. for allowing us to conduct our studies.
We also thank our friends of the class of Herpetology
2012-1: Katherin Linares, Stefanny Barros, Ricardo
Martinez, and Karen Vega and also to our friends of the
Herpetology Lab of Magdalena University: Danny Ver-
gara, Juan Jimenez, Efram Rada, Miguel Arevalo, Mar-
tin Caicedo, Heman Granda Rodriguez, Carlos Villa de
Leon, Liliana Saboya, Danilo Vergara, and Caitlin Webb
(and for reviewing the manuscript). Special mention goes
to colleagues John D. Lynch, Julio Mario Hoyos, Cesar
Barrio Amoros, German Lorero Medina, Victor Acosta
Chaves, Andres R. Acosta Galvis, Paulo Tigreros, and
Luis Duarte and for their contributions to the manuscript.
Linally, we thank the Centro de Colecciones Biologicas
de la Universidad del Magdalena for their support and
protection of all our vouchers.
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sphere Reserve of Veracruz, Mexico. Biological Con-
servation 132(1): 61-75.
Urbina-Cardona JN, Londono-Murcia MC, Garcia- Avila
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do de alteracion antropogenica en el Parque Nacional
Natural Isla Gorgona, Pacifico colombiano. Caldasia
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Thorson TB. 1955. The relationship of water economy
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Whitfield SM, Reider K, Greenspane S, Donelly MA.
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A natural history resume of native populations. Smith-
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Oscar Angarina-M. is an undergraduate biology student at Universidad del Magdalena, Santa Marta, Colombia.
His interests are in the study of herpetofaunal community ecology, habitat fragmentation, and environment legisla-
tion.
Andres Camilo Montes-Correa is a biology student at Universidad del Magdalena, Santa Marta, Colombia. Since
its inception, be joined the Herpetology Lab, where he began to develop interest in ecological, taxonomic, and
systematic studies. Among his current research projects are feeding ecology of leaf-litter tropical dry forest lizards,
taxonomy of Caribbean Dwarf Geckos (Lepidoblepharis), and habitat use of Orinoquian Freshwater Turtles (chelids
and kinosternids).
Juan Manuel Renjifo is a Colombian herpetologist, wildlife photographer, and biologist at Pontificia Universi-
dad Javeriana, having received his M.Sc. degree at the University of Kansas. He has developed studies in ecology
and taxonomy of Colombian herpetofauna, ophidism, and snakebite. Juan has served as director of Laboratorio de
Sueros Antioffdicos of Instituto Nacional de Salud for 30 years and is dedicated to teaching (ad honorem) at the
Universidad Nacional de Colombia and Universidad del Magdalena universities.
April 2015 | Volume 8 | Number 1
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Herpetofauna of an agroforestry system in the Colombian Caribbean
Appendix I. Amphibian Caribbean lowlands inventories used for Bray-Curtis Similarity Analyses. A = La Gloria Project; B =
El Botillero (Duenez-Gomez et al. 2004); C = Ensenada Neguanje (Rueda-Solano and Castellanos-Barliza 2010); D = Medio
Rancheria (Blanco-Torres et al. 2013); E = Reserva Eorestal Protectora Montes de Oca (Galvis et al. 2011); E = Serrama de Coraza
(Galvan-Guevara and De la Ossa- Velasquez 2011); G = los Montes de Maria y la Cienaga La Caimanera (Acosta-Galvis 2012b);
H = Represa de Urra (Renjifo and Lundberg 1999); I = Murrucucu lowlands (sensu Romero-Martmez et al. 2008); J = Humedales
del Cordoba (Romero-Martmez and Lynch 2010); K = Atlantico and North Bolivar (Cuentas et al. 2002); L = Santuario de Vida
Silvestre Los Besotes (Rueda-Almonacid et al. 2008a); M = Cienaga del Zapatosa (Medina-Rangel et al. 2011); N = Universidad
del Magdalena (Montes-Correa et al. 2015).
Species
A
B
c
D
E
F
G
H
1
J
K
L
M
N
Rhinella humboldti
1
1
0
1
1
1
1
1
0
1
1
1
1
1
Rhinella margaritifera
0
0
0
0
0
0
1
0
1
0
0
0
0
0
Rhinella marina
1
1
1
1
1
1
1
1
1
1
1
1
1
1
Rhinella sternosignata
0
0
0
0
1
0
0
0
0
0
0
0
0
0
Rhaebo haematiticus
0
0
0
0
0
1
0
1
0
0
0
0
0
0
Hyalinobatrachium collymbiphyllum
0
0
0
0
0
0
0
1
0
0
0
0
0
0
Hyalinobatrachium fleischmanni
0
0
0
0
0
0
1
1
0
0
0
0
0
0
Ceratophrys calcarata
1
1
0
1
1
1
1
1
0
1
1
1
1
0
Craugastor raniformis
0
0
0
0
0
0
1
1
1
1
1
0
1
0
Pristimantis taeniatus
0
0
0
0
0
0
0
0
1
0
0
0
0
0
Pristimantis viejas
0
0
0
0
0
0
0
0
1
0
0
0
0
0
Strabomantis bufoniformis
0
0
0
0
0
0
0
1
0
0
0
0
0
0
Eleutherodactylus johnstonei
0
0
0
0
0
0
0
0
0
0
1
0
0
0
Colostethus pratti
0
0
0
0
0
0
0
0
1
0
0
0
0
0
Colostethus ruthveni
0
0
1
0
0
0
0
0
0
0
0
0
0
0
Dendrobates truncatus
0
0
1
0
0
1
1
1
1
1
1
0
1
0
Cryptobatrachus boulengeri
0
0
1
0
0
0
0
0
0
0
0
0
0
0
Dendropsophus ebraccatus
1
0
0
0
0
0
0
1
0
1
0
0
0
0
Dendropsophus microcephalus
1
1
0
1
1
1
1
1
0
1
1
0
1
0
Hypsiboas boans
0
0
0
0
0
1
0
1
1
0
1
0
1
0
Hypsiboas crepitans
1
1
1
0
1
0
1
0
0
1
1
1
1
0
Hypsiboas pugnax
1
0
1
1
1
1
1
1
1
1
1
1
1
1
Hypsiboas rosenbergi
0
0
0
0
0
0
0
0
1
0
0
0
0
0
Phyllomedusa venusta
1
1
0
0
0
0
1
1
1
0
1
1
0
0
Pseudis paradoxa
1
1
0
0
0
0
1
1
0
1
1
0
0
0
Scarthyla vigilans
1
1
0
1
0
1
1
1
0
1
1
0
0
0
Scinax boulengeri
0
0
0
0
0
0
0
1
0
0
1
0
0
0
Scinax elaeochrous
0
0
0
0
0
0
0
1
0
0
0
0
0
0
Scinax rostratus
1
1
0
0
0
0
1
0
0
1
0
0
1
0
Scinax ruber
0
1
0
1
1
1
1
1
1
1
1
0
0
0
Scinax x-signatus
1
0
0
0
0
0
0
0
0
1
0
0
0
0
Smilisca phaeota
0
0
0
0
0
0
0
1
1
0
0
0
0
0
Smilisca sila
0
0
0
0
0
0
0
1
0
0
0
0
0
0
Trachycephalus typhonius
1
0
0
1
1
1
1
1
0
1
1
0
1
0
Engystomops pustulosus
1
1
1
1
1
0
1
1
1
1
1
1
1
1
Pleurodema brachyops
1
1
1
1
1
0
1
1
0
1
1
1
1
1
Pseudopaludicola pusilla
1
1
0
1
1
0
1
1
0
1
1
0
1
0
Leptodactylus colombiensis
0
0
0
0
1
0
0
0
0
0
0
0
0
0
Leptodactylus fragilis
1
1
1
1
0
0
1
1
0
1
1
0
1
0
Leptodactylus fuscus
1
1
0
1
1
1
1
1
0
1
1
1
1
1
Leptodactylus insularum
1
1
1
1
1
1
1
1
0
1
1
1
1
1
Leptodactylus poecilochilus
1
1
0
1
1
0
1
0
1
1
1
1
1
0
April 2015 | Volume 8 | Number 1
Amphib. Reptile Conserv.
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Angarita-M et al.
Appendix I (Continued). Amphibian Caribbean lowlands inventories used for Bray-Curtis Similarity Analyses. A = La Gloria
Project; B = El Botillero (Duenez-Gomez et al. 2004); C = Ensenada Neguanje (Rueda-Solano and Castellanos-Barliza 2010); D
= Medio Rancheria (Blanco-Torres et al. 2013); E = Reserva Eorestal Protectora Montes de Oca (Galvis et al. 2011); E = Serrama
de Coraza (Galvan-Guevara and De la Ossa- Velasquez 2011); G = los Montes de Maria y la Cienaga La Caimanera (Acosta-Galvis
2012b); H = Represa de Urra (Renjifo and Lundberg 1999); I = Murrucucu lowlands (sensu Romero-Martmez et al. 2008); J =
Humedales del Cordoba (Romero-Martmez and Lynch 2010); K = Atlantico and North Bolivar (Cuentas et al. 2002); L = Santuario
de Vida Silvestre Los Besotes (Rueda-Almonacid et al. 2008a); M = Cienaga del Zapatosa (Medina-Rangel et al. 2011); N = Uni-
versidad del Magdalena (Montes-Correa et al. 2015).
Species
A
B
c
D
E
F
G
H
1
J
K
L
M
N
Leptodactylus savagei
0
0
0
0
0
0
0
1
1
0
1
0
0
0
Lithodites lineatus
0
0
0
0
1
0
0
0
0
0
0
0
0
0
Elachistocleis panamensis
1
0
1
1
1
0
0
1
0
1
1
0
1
0
Elachistocleis pearsei
1
0
0
0
0
1
1
1
0
1
1
0
1
0
Pipa parva
0
0
0
1
1
0
0
0
0
0
0
0
0
0
Lithobates vaillanti
0
0
0
0
1
0
0
1
0
0
1
1
1
0
Caecilia isthmica
0
0
0
0
0
0
1
0
0
0
0
0
0
0
Caecilia caribea
1
0
0
0
0
0
0
0
0
0
1
0
0
0
Caecilia subnigricans
0
0
0
0
0
0
0
1
0
1
0
0
0
0
Oscaecilia polyzona
0
0
0
0
0
0
0
1
0
0
0
0
0
0
Typhlonectes natans
0
1
0
0
0
1
1
1
0
1
0
0
0
0
Bolitoglossa biseriata
0
0
0
0
0
1
0
1
1
0
0
0
0
0
April 2015 | Volume 8 | Number 1
Amphib. Reptile Conserv.
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Herpetofauna of an agroforestry system in the Colombian Caribbean
Appendix II. Reptile Caribbean lowlands inventories used for Bray-Curtis Similarity Analyses. A = La Gloria Project; B = El Botil-
lero (Duenez-Gomez et al. 2004); C = Ensenada Neguanje (Rueda-Solano and Castellanos-Barliza 2010); D = Medio Rancheria
(Blanco-Torres et al. 2013); E = Reserva Eorestal Protectora Montes de Oca (Galvis et al. 2011); E = Serrama de Coraza (Galvan-
Guevara and De la Ossa- Velasquez 2011); G = Represa de Urra (Renjifo and Lundberg 1999); H = Humedales del Cordoba (Car-
vajal-Cogollo et al. 2007); I = Santuario de Vida Silvestre Los Besotes (Rueda-Almonacid et al. 2008b); J = Cienaga del Zapatosa
(Medina-Rangel et al. 2011); K = Universidad del Magdalena (Montes-Correa et al. 2015).
Species
A
B
c
D
E
F
G
H
1
J
K
Amphisbaena alba
0
0
0
0
1
0
0
0
1
0
0
Amphisbaena fuliginosa
0
0
0
0
1
0
0
0
1
0
0
Amphisbaena medemi
0
0
0
1
0
0
0
0
1
0
0
Gonatodes albogularis
1
1
1
1
1
1
1
1
1
1
1
Gonatodes vittatus
0
0
0
1
1
0
0
0
0
0
0
Lepidoblepharis sanctaemartae
1
0
1
1
1
0
0
1
1
1
1
Sphaerodactylus heliconiae
0
0
0
0
0
0
0
0
0
1
0
Phyllodactylus ventralis
0
0
1
1
1
0
0
0
2
0
1
Thecadactylus rapicauda
1
1
1
1
1
0
1
1
0
1
1
Hemidactylus brookii
0
0
1
1
1
1
1
1
1
0
1
Hemidactylus frenatus
1
0
0
0
1
0
0
0
0
1
1
Basiliscus basilicus
1
1
0
1
1
1
1
1
0
1
0
Basiliscus galeritus
0
0
0
0
0
0
0
1
0
0
0
Corytophanes cristatus
0
0
0
0
0
0
1
0
0
0
0
Anolis auratus
1
1
1
1
1
1
1
1
1
1
1
Anolis biporcatus
0
0
0
0
1
0
0
0
0
0
0
Anolis pentaprion
0
0
0
0
0
0
1
0
0
0
0
Anolis onca
0
0
0
0
1
0
0
0
0
0
0
Anolis tropidogaster
0
0
0
0
1
0
1
1
0
1
0
Anolis vittigerus
0
0
0
0
0
1
0
1
0
0
0
Iguana iguana
1
1
1
1
1
1
1
1
1
1
1
Polychrus marmoratus
0
0
1
1
1
0
0
0
1
1
0
Stenocercus erythrogaster
0
0
1
0
1
0
0
0
1
1
0
Maracaiba zuliae
1
0
0
0
1
0
0
0
0
0
0
Mabuya sp.
0
1
1
1
0
1
1
1
1
1
0
Bachia bicolor
0
0
1
0
0
0
0
0
0
1
1
Bachia talpa
0
0
0
1
1
0
0
0
1
0
0
Gymnophthalmus speciosus
0
0
0
1
1
0
0
1
1
1
1
Leposomoma rugiceps
1
1
1
0
0
0
1
1
0
1
0
Tretioscincus bifasciatus
1
1
1
1
1
0
0
1
1
1
1
Ameiva praesignis
1
1
1
1
1
1
1
1
1
1
1
Ameiva bifrontata
1
0
1
1
1
0
0
0
0
0
1
Cnemidophorus lemniscatus
1
1
1
1
1
1
1
1
1
1
1
Holcosus festivus
0
0
0
0
0
1
1
1
0
1
0
Tupinambis teguixin
0
1
0
1
1
1
1
1
1
1
0
Liotyphlops albirostris
1
0
1
1
1
1
1
1
0
0
1
Epictia goudotii
0
0
0
1
0
0
0
0
1
0
1
Trilepida macrolepis
0
0
0
0
0
0
1
0
0
0
0
Trilepida dugandi
0
0
0
0
0
0
0
0
1
0
0
Boa constrictor
1
1
1
1
1
1
1
1
1
1
1
Corallus batesi
0
0
0
0
0
0
1
1
0
0
0
Corallus ruschenbergerii
0
0
1
1
1
1
1
1
0
1
0
Epicrates maurus
1
1
0
1
1
1
1
1
0
1
0
Chironius carinatus
1
0
0
0
1
1
1
1
0
1
0
Amphib. Reptile Conserv.
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April 2015 1
Volume 8 |
Number 1
1 e92
Angarita-M et al.
Appendix II (continued). Reptile Caribbean lowlands inventories used for Bray-Curtis Similarity Analyses. A = La Gloria Project;
B = El Botillero (Duenez-Gomez et al. 2004); C = Ensenada Neguanje (Rueda-Solano and Castellanos-Barliza 2010); D = Medio
Rancberia (Blanco-Torres et al. 2013); E = Reserva Eorestal Protectora Montes de Oca (Galvis et al. 2011); E = Serrama de Coraza
(Galvan-Guevara and De la Ossa- Velasquez 2011); G = Represa de Urra (Renjifo and Lundberg 1999); H = Humedales del Cordoba
(Carvajal-Cogollo et al. 2007); I = Santuario de Vida Silvestre Los Besotes (Rueda-Almonacid et al. 2008b); J = Cienaga del Zapa-
tosa (Medina-Rangel et al. 2011); K = Universidad del Magdalena (Montes-Correa et al. 2015).
Species
A
B
c
D
E
F
G
H
1
J
K
Coluber mentovarius
0
0
0
0
1
0
0
0
0
0
0
Dendrophidion bivittatus
0
0
0
0
0
1
1
0
0
0
0
Dendrophidion percarinatus
0
0
0
0
0
0
1
0
0
0
0
Drymarchon caudomaculatus
0
0
0
1
0
0
0
0
0
0
0
Drymarchon melanurus
0
0
0
0
1
0
0
0
1
0
0
Leptophis ahaetulla
1
0
0
1
1
1
0
1
1
1
0
Mastigodryas boddaertii
0
0
0
1
1
0
1
0
1
0
1
Mastigodryas pleei
0
1
1
1
1
0
1
1
1
1
0
Oxybelis aeneus
0
1
1
1
1
1
1
0
0
1
1
Oxy bells fulgidus
0
0
1
0
1
0
0
0
1
0
0
Pliocercus euryzonus
0
0
0
0
0
0
1
0
0
0
0
Pseustes poecilonotus
0
0
0
0
0
0
1
0
0
0
0
Pseustes shropshieri
0
0
0
0
0
0
1
0
0
0
0
Spillotes pullatus
0
1
0
1
0
1
1
1
1
1
0
Stenorrhina degenhardtii
0
0
0
0
0
0
1
0
0
0
0
Tantilla melanocephala
1
0
0
0
1
0
0
1
1
0
1
Tantilla semicincta
0
0
0
1
1
0
0
0
1
0
1
Clelia Clelia
0
0
0
0
1
1
1
1
1
0
0
Enulius flavitorques
0
0
0
0
1
0
0
1
1
1
1
Erythrolamprus melanotus
0
0
0
1
1
0
1
1
1
0
0
Erythrolamprus bizona
0
0
0
0
0
0
0
0
1
0
0
Helicops danieli
1
1
1
0
0
1
1
1
0
1
0
Imantodes cenchoa
1
0
0
0
1
1
1
0
1
1
0
Leptodeira annulata
1
0
1
1
1
0
0
1
1
0
1
Leptodeira septentrionalis
1
1
0
0
0
1
0
0
0
1
1
Lygophis lineatus
1
0
0
0
1
1
1
1
1
1
0
Ninia atrata
0
0
0
0
0
0
1
0
0
0
0
Oxyrhopus petolarius
1
0
0
0
1
0
1
0
0
0
0
Phimophis guianensis
0
1
1
1
1
0
0
0
1
1
1
Pseudoboa neuwiedii
1
1
1
1
1
0
1
1
0
1
0
Sibon nebulatus
0
0
0
0
1
0
1
0
0
0
0
Thamnodynastes paraguanae
1
0
0
1
1
0
0
0
0
0
0
Thamnodynastes gambotensis
1
1
0
0
0
0
1
1
0
1
0
Xenodon severus
0
0
0
1
1
0
0
0
0
0
0
Xenodon rabdocephalus
1
0
0
0
1
0
0
0
0
0
0
Micrurus camilae
0
0
0
0
0
0
1
0
0
0
0
Micrurus dissoleucus
1
0
0
1
1
0
1
0
1
0
1
Micrurus dumerili
0
0
0
0
1
0
0
1
1
0
0
Bothriechis schlegelii
0
0
0
0
0
0
1
0
0
0
0
Bothrops asper
1
1
1
0
1
0
1
1
1
1
0
Crotalus durissus
1
0
1
1
1
0
0
0
1
1
1
Porthidium lansbergii
1
1
1
1
1
0
0
1
1
1
1
Porthidium nasutum
0
0
0
0
0
0
1
0
0
0
0
Mesoclemmys dahli
1
0
0
0
0
0
0
1
0
1
0
Amphib. Reptile Conserv.
161
April 2015 1
Volume 8 |
Number 1
1 e92
Herpetofauna of an agroforestry system in the Colombian Caribbean
Appendix II (continued). Reptile Caribbean lowlands inventories used for Bray-Curtis Similarity Analyses. A = La Gloria Project;
B = El Botillero (Duenez-Gomez et al. 2004); C = Ensenada Neguanje (Rueda-Solano and Castellanos-Barliza 2010); D = Medio
Rancberia (Blanco-Torres et al. 2013); E = Reserva Eorestal Protectora Montes de Oca (Galvis et al. 2011); E = Serrama de Coraza
(Galvan-Guevara and De la Ossa- Velasquez 2011); G = Represa de Urra (Renjifo and Lundberg 1999); H = Humedales del Cordoba
(Carvajal-Cogollo et al. 2007); I = Santuario de Vida Silvestre Los Besotes (Rueda-Almonacid et al. 2008b); J = Cienaga del Zapa-
tosa (Medina-Rangel et al. 2011); K = Universidad del Magdalena (Montes-Correa et al. 2015).
Species
A
B
c
D
E
F
G
H
1
J
K
Podocnemis lewyana
0
0
0
0
0
0
1
0
0
1
1
Chelydra acutirostris
0
0
0
0
0
0
1
0
0
0
0
Cryptochelys leucostomum
0
0
0
0
1
0
1
0
0
0
0
Kinosternon scorpioides
0
0
0
0
1
0
1
1
1
1
1
Rhinoclemmys melanosterna
0
0
0
0
1
0
0
1
0
1
0
Trachemys calliwstris
1
1
0
1
1
0
1
1
0
1
1
Chelonoidis carbonaria
1
1
0
1
1
0
1
1
1
1
1
Crocodylus acutus
0
0
0
0
1
0
1
0
0
1
0
April 2015 | Volume 8 | Number 1
Amphib. Reptile Conserv.
162
e92
Copyright: © 2014 Pierson et al. This is an open-access article distributed under
the terms of the Creative Commons Attribution-NonCommercial-NoDerivs 3.0
Unported License, which permits unrestricted use for non-commercial and educa-
tion purposes only provided the original author and source are credited. The of-
ficial publication credit source: Amphibian & Reptile Conservation at: amphibian-
reptile-conservation. org
Amphibian & Reptiie Conservation 8(1) [Gen Sec]: 1-6.
A survey for the Chinese giant salamander {Andrias
davidianus', Blanchard, 1871) in the Qinghai Province
Todd W. Pierson, ^yan Fang, ^WANG Yunyu, and Theodore Papenfuss
^University of Georgia, 150 East Green Street, Athens, Georgia, 30602, USA ^State Key Laboratory of Genetic Resources and Evolution, and Yun-
nan Laboratory of Molecular Biology of Domestic Animals, Kunming Institute of Zoology, Chinese Academy of Sciences, 32 Jiaochang Donglu,
Kunming, Yunnan, 650223, CHINA ^Museum of Vertebrate Zoology, University of California, Berkeley, California, 94720, USA
Abstract . — ^The Chinese giant saiamander {Andrias davidianus) was once common, but it has de-
ciined precipitousiy in the past severai decades. An enigmatic specimen coiiected in 1966 repre-
sents the oniy historicai record of the species from the Qinghai-Tibetan Piateau. From June-Juiy
2012, we conducted opportunistic community inquiries and fieid surveys in Qinghai to attempt to
iocate Andrias. We received anecdotai evidence that additionai Andrias have been found in recent
years, but we faiied to discover any Andrias during our fieid surveys. We suspect that reiict popuia-
tions persist in Qinghai, but the significant degradation of stream quaiity in the region iikeiy threat-
ens the iong-term survivai of any remaining Andrias. Here, we provide a brief overview of Andrias
conservation, a summary of our surveys, and emphasize the importance of continued searches for
this geographicaiiy disjunct popuiation.
Key words. Cryptobranchidae, Qinghai-Tibetan Plateau, conservation
Citation: Pierson TW, Van F, Wang Y, Papenfuss T. 2014. A survey for the Chinese giant salamander {Andrias davidianus] Blanchard, 1871) in the Qing-
hai Province. Amphibian & Reptile Conservation 8{t) [General Section]; 1-6 (e74).
Introduction
The Chinese Giant Salamander {Andrias davidianus) was
once widely distributed throughout the Yangtze, Yellow,
and Pearl River drainages. However, dramatic declines
since 1950 have restricted the species to twelve frag-
mented regions across seventeen provinces (Zhang et al.
2002). These declines are due largely to habitat degrada-
tion and harvest for food (Dai et al. 2009). In response to
these declines in the wild, the 2004 International Union
for Conservation of Nature Red List evaluated A. davidi-
anus as Critically Endangered, and the recognition of the
conservation needs of the species has attracted national
and international attention. Additionally, at least thirty
preserves have been established in China to conserve A.
davidianus, and captive breeding for human consump-
tion has increased in prevalence and success (Dai et al.
2009; Zhang et al. 2002). One challenge for the conser-
vation of A. davidianus is the preservation of genetic
diversity, and several studies have examined variation
between and among populations of A. davidianus. Sig-
nificant substructuring exists among populations (Mur-
phy et al. 2000; Tao et al. 2006), although results may
be confounded by translocations of animals through the
food trade. However, the overall genetic diversity of A.
davidianus is relatively low compared to other salaman-
ders (Tao et al. 2005; Yang et al. 2011).
A single specimen of A. davidianus was collected in
the headwaters of the Yangtze River in the Qinghai Prov-
ince in August 1966 (33.898 96.522; Fig. 1; Trap Loca-
tion 9, Figure 2; Fig. 3). The specimen was a gravid fe-
male caught on hook-and-line near the town of Bagan at
approximately 4,200 m, representing the highest known
distribution record of A. davidianus by more than 2,000
m and a greatly disjunct population (Chen 1989). The ge-
ography and the geological history of this region (Yin
2010) suggest the possibility that the gap between this
Qinghai record and other known localities for A. davidi-
anus represents a true biogeographical break, and this
population may be important for conservation purposes.
From 6 June to 2 July 2012, we used a variety of
methods to survey Qinghai for A. davidianus. We were
unsuccessful in locating any Andrias, but here we report
the environmental conditions of the historic locality and
others, anecdotal reports of Andrias from locals, and sug-
gestions for future efforts to locate Andrias in Qinghai.
Methods
Throughout our stay in Qinghai, we frequently talked to
officials from the Bureau of Forestry to obtain permis-
sion to search for Andrias. During this process, we also
inquired about anecdotal Andrias sightings from fisher-
men. This amounted to discussions with approximately
Correspondence, ^twpierso® uga.edu; twpierson® gmail.com
Amphib. Reptile Conserv. | amphibian-reptile-conservation.org (1)
January 2014 | Volume 8 | Number 1 | e74
Pierson et al.
fifteen government officials and five local fishermen. Af-
ter talking with government officials and locals of Qin-
ghai, we selected sites to survey based on historical and
anecdotal records. We trapped in three general regions —
Bagan, Zhiduo, and Yushu (Table 1; Fig. 2) — including
the exact locality of the collection of the 1966 specimen
from Bagan (Trap Location 9, Figure 2).
Browne et al. (2011) reviewed and evaluated survey
techniques for cryptobranchid salamanders. Because of
religious and cultural beliefs about the sanctity of fish,
local people in Qinghai are overwhelmingly unsupport-
ive of any attempts by biologists to survey aquatic or-
ganisms. Due to these limitations, some methods (e.g.,
electroshocking) were not possible, and our field surveys
were conducted primarily through trapping, which has
been shown to be reasonably effective for surveying for
Cryptobranchus a. alleganiensis and A. japonicus (Fos-
ter et al. 2008; Briggler et al. 2013). Even so, we were
restricted to trapping discretely, had several traps stolen,
and were actively discouraged from actually entering the
streams by both locals and governmental officials. These
practical challenges significantly limited our trapping
efforts. We primarily used two sizes of custom-made,
mesh-net rectangular crab traps (approximately 81 x 61
X 28 cm; 61 x 46 x 20 cm) designed to catch Andrias of
varying sizes. The traps were baited altematingly with
sardines, fishmeal, liver, and sponges soaked in fish oil
Fig. 1. The adult female Andrias captured in Qinghai, China
in 1966. This specimen now resides at the Northwest Plateau
Institute of Biology in Xining.
held in bait containers. Traps were weighted with stones,
anchored to shore, and entirely submerged in 0.3 - >5
m of water in suitable habitat. Typically, the traps were
placed in still pools along rocky bluffs at the edge of the
river and checked after approximately 24 hours. Addi-
tionally, baited hook-and-line and manual searches of
rocky habitat were used opportunistically when the habi-
tat was suitable.
Results and Discussion
During our discussions with local people and govern-
ment officials, we heard several anecdotal reports of
Andrias being caught in recent years. Local Bureau of
Forestry officials and one layman in Qumalai told of an
adult Andrias that had been caught and thrown back by
a fisherman at the same locality as the original record
(Trap Locality 9, Figure 2) around 1992. The same of-
ficials in Qumalai and several officials in Zhiduo told of
an Andrias that had been caught in the Nieqia River at its
confluence with the Tongtian River in Qumalai (34.016,
95.817) between 1996-1997. This individual was re-
portedly sent to Xian and sold for food. An official from
Zhiduo also reported that this fisherman’s brother had
caught an Andrias in a slow part of the Tongtian River
between Zhiduo and Yushu earlier in 2012. Finally, two
residents of Yushu reported seeing dead Andrias in the
Tongtian River after the earthquake of 2010. Only one
other species of caudate (Batrachuperus tibetanus) is
Table 1. Trapping effort in Qinghai. Numbers to right of the
location indicate the corresponding points on Fig. 2.
Date Placed
Traps
Location
13 June
12
Four tributaries of De Qu River near
Bagan (1^)
13 June
4
De Qu River on the road to Bagan (5)
14 June
5
Bo Qu River near Bagan (6-8)
14 June
4
De Qu River at the bridge in Bagan (9)
15 June
12
Four tributaries of De Qu River near
Bagan (1-4)
15 June
1
De Qu River on the road to Bagan (5)
16 June
12
Four tributaries of De Qu River near
Bagan (1-4)
16 June
5
Upper De Qu River outside of Bagan
17 June
5
De Qu River at the bridge in Bagan (9)
19 June
A
Tribuatries of Hie Qu River near Zhiduo
(10-11)
20 June
14
Tribuatries of Hie Qu River near Zhiduo
(10-11)
21 June
14
Tribuatries of Hie Qu River near Zhiduo
(10-11)
26 June
10
Tributaries of Tongtian River near Yushu
(12)
28 June
14
Tributaries of Tongtian River near Yushu
(12)
TOTAL
116
Amphib. Reptile Conserv. | amphibian-reptile-conservation.org (2) January 2014 | Volume 8 | Number 1 | e74
Andrias davidianus survey in Qinghai Province, China
Fig. 2. Map of trapping localities and nearby towns in Qinghai.
present in the region, but its limited distribution in Qing-
hai and small size make it unlikely to have been misiden-
tified as Andrias in locals’ reports. Although there is no
hard evidence to substantiate the reports we heard, when
taken in aggregate, they seem credible.
We trapped for 106 trap-nights (Table 1) and were
not able to discover any Andrias during our field survey
of Qinghai. Foster et al. (2008) used a similar trapping
protocol and caught Cryptobranchus a. alleganiensis at
a rate of 0.01-0.10 captures/trap-night. Briggler et al.
(2013) trapped for C. a. alleganiensis in deeper and more
turbid water and reported an average capture rate of 0.042
captures/trap-night with net-mesh traps. We acknowl-
edge that our limited number of trap-nights prevents us
from making definitive conclusions about the presence or
absence of A. davidianus at our trapping sites.
Virtually all of the streams in which we trapped were
turbid and swollen with silted water (Fig. 3), which is a
major threat to Andrias conservation. While it is possible
that some of this turbidity was due to seasonal snowmelt,
it is more likely that anthropogenic causes are primarily
responsible. Since the collection of the lone specimen in
1966, mining for gold and other valuable commodities
has become prevalent throughout the Qinghai-Tibetan
Plateau. Furthermore, dozens of active sand and gravel
mining operations were stationed throughout the rivers
we sampled (Fig. 4). Locals in Yushu reported an in-
crease in mining activity in response to construction and
reparation needs following the major earthquake of 2010.
Additionally, some streamside microhabitats for Andrias
have been degraded due to road and bridge construction
(Fig. 5). Another contributor to the siltation of Qinghai
streams may be grassland degradation and desertification
driven by climate change that has been demonstrated in
the region (Cui and Graf 2009).
Conclusion
Despite our inability to locate Andrias in Qinghai, anec-
dotal reports suggest that relict populations may still exist
throughout the former range of the species. However, the
apparent dramatic declines in stream quality in the region
probably threaten the persistence of these populations.
Although more remote regions further west of Bagan
have fewer roads and present more practical challenges
to fieldwork, they hold large headwaters of the Yangtze
upstream of significant mining activity and may repre-
sent the most suitable remaining habitat. While we were
not able to survey these regions during our expedition,
they should be prioritized in future searches. Because
Qinghai is at such a high elevation, suitable conditions
for searching occur in a small window each year. We rec-
onnnend that efforts be focused in August or September,
after seasonal flooding from snowmelt has passed, but
before winter has returned. In addition to the continued
use of trapping, hook-and-line, and manual searches, we
recommend the possibility of using environmental DNA,
which has been demonstrated to be an effective tool for
detecting populations of other cryptobranchids (e.g., Ol-
son et al. 2012; S. Spear, pers. comm.).
Because of the potential importance of this geographi-
cally isolated population of Andrias in Qinghai, its redis-
Amphib. Reptile Conserv. | amphibian-reptile-conservation.org (3)
January 2014 | Volume 8 | Number 1 | e74
Pierson et al.
Fig. 3. The locality where the first and only Andrias was collected from Bagan, Qinghai in 1966. Today, the water is turbid and ap-
pears largely unsuitable for Andrias.
Fig. 4. A mining operation on the banks of the Tongtian River, near Qumalai, Qinghai.
Amphib. Reptile Conserv. | amphibian-reptile-conservation.org (4)
January 2014 | Volume 8 | Number 1 | e74
Andrias davidianus survey in Qinghai Province, China
Fig. 5. Stream bank degradation caused by road construction along the Tongtian River.
covery should continue to be a top priority for Andrias
conservation.
Acknowledgments. — Funding was provided by
the Key Program of the Chinese Academy of Sciences
(XDB03030000), the Strategic Priority Research Pro-
gram of the Chinese Academy of Sciences (KJZD-EW-
L07), the National Geographic Society’s Young Explor-
ers Grant (9084-12), the Explorers Club Youth Activity
Fund, the California Academy of Sciences, and the Kun-
ming Institute of Zoology. We would like to thank J. Che
for her great help in planning and organizing the trip and
Y. Zhang for aid in acquiring permission to conduct re-
search in Qinghai. Additionally, the Chinese Academy of
Sciences (including the Northwest Institute of Biology)
and local forestry departments proved important for the
success of the expedition.
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Received: 03 December 2013
Accepted: 17 January 2014
Published: 22 January 2014
Todd Pierson’s research focuses on amphibian and reptiles ecology, evolution, and conservation. He grad-
uated with a B.S. Ecology from the Odum School of Ecology at the University of Georgia in 2013. He
currently works in the EHS DNA Lab at UGA, where be develops environmental DNA assays for use in
detecting aquatic amphibians.
Yan Fang is mainly interested in tbe pbylogeography and conservation genetics of amphibians. She gradu-
ated with a Ph.D. from the Kunming Institute of Zoology (KIZ), Chinese Academy of Sciences in 2013.
Now she works on the conservation genetics of Chinese giant salamander at KIZ.
Wang Yunyu is staff at the Southern China DNA Barcoding Center (SCDBC), Kunming Institute of Zool-
ogy^ Chinese Academy of Science. She currently works on DNA barcoding of amphibians and reptiles.
Theodore Papenfuss is a Research Scientist at the Museum of Vertebrate Zoology. His current research
activities involve field studies of amphibians and reptiles in Asia and Central America. He is also collabo-
rating with conservation agencies that are conducting surveys of tropical forests in Guatemala in order to
select areas for permanent habitat protection.
Amphib. Reptile Conserv. | amphibian-reptile-conservation.org (6)
January 2014 | Volume 8 | Number 1 | e74
Copyright: © 2014 Michaels et al. This is an open-access article distributed under the
terms of the Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported
License, which permits unrestricted use for non-commercial and education purposes only
provided the original author and source are credited. The official publication credit source:
Amphibian & Reptile Conservation at: amphibian-reptile-conservation.org
Amphibian & Reptiie Conservation
[General Section] 8(1) :7-23.
REVIEW
The importance of enrichment for advancing amphibian
welfare and conservation goals: A review of a neglected topic
^Christopher J. Michaels , Roger Downie, and ^Roisin Campbell-Palmer
^•‘^Preziosi Group, Faculty of Life Sciences, Michael Smith Building, University of Manchester, Manchester, ENGLAND ^School of Life Sciences,
Graham Kerr Building, University of Glasgow, Glasgow, SCOTLAND ^Conservation Programmes, The Royal Zoological Society of Scotland,
Edinburgh, SCOTLAND
Abstract. — Enrichment, broadiy the provision of stimuii to improve the weifare of captive animais,
is known to be important in husbandry practice and in the success of ex situ conservation and
reintroduction programs. Practicai evidence of the importance of enrichment exists for a number of
taxa, yet amphibians are pooriy represented. There is no reason to assume a priori that amphibians
wouid not benefit from enrichment and, given their increasing prominence in captive programs,
their requirements in captivity beyond basic husbandry shouid be the focus of more intense
study. We review the existing body of research on enrichment for amphibians, as weii as that
for fish and reptiies, which may be regarded as behavioraiiy and neuroiogicaiiy broadiy simiiar
to amphibians. We aiso briefiy discuss mechanisms by which enrichment might affect amphibian
fitness and, therefore, reintroduction success. Our review supports the contention that there may be
important consequences of enrichment for both captive weifare and ex situ conservation success
in amphibians and that amphibian enrichment effects may be highiy variabie taxonomicaiiy. In the
face of increasing numbers of captive amphibian species and the importance of exs/fupopuiations
in ensuring their species ievei persistence, enrichment for amphibians may be an increasingiy
important research area.
Key words. Behavior, conservation, environmental enrichment, re-introduction, welfare, ex situ, fish, reptiles
Citation: Michaels CJ, Downie JR, Campbell-Palmer R. 2014. The importance of enrichment for advancing amphibian welfare and conservation goals:
A review of a neglected topic. Amphibian & Reptiie Conservation 8(1) [General Section]: 7-23 (e77).
Introduction
A wide range of amphibian species is currently main-
tained in captivity. Some species are used as models in
laboratory research, including the ubiquitous Xenopus
laevis and the dendrobatid frogs used to study skin pep-
tides (reviewed by Daly 1998) and caecilians used in bio-
mechanics research (e.g., Summers and O’Reilly 1997)
and leaf frogs involved in conservation research (Ogilvy
et al. 2012a, b). Several species are farmed (in addition
to the many collected from the wild) for food or other
products and others are maintained by private individuals
as hobby or pet animals (Gascon et al. 2005). In addition,
the ex situ conservation response to the on-going global
amphibian extinction crisis (e.g., Gagliardo et al. 2008;
Lee et al. 2006; Norris 2007) has drawn much public-
ity to the growing number of amphibians maintained for
conservation breeding and education in zoos and similar
institutions. This increase in captive amphibians (both
in actual numbers and species held) and their mounting
conservation importance, has highlighted the need for
a more thorough understanding of amphibian captive
husbandry (Gascon et al. 2005), particularly for species
that have no history in captivity and for those that are
intended for release into the wild (Gagliardo et al. 2008;
Gascon et al. 2005).
For many other taxa, the importance of enrichment
has been identified for not only the welfare, or the physi-
cal and psychological wellbeing, of individual animals
in captivity or those destined for release, but also for
the overall/long-term success of reintroduction projects
(Crane and Mathis 2010; Shepherdson et al. 1998; Young
2003). However, the implications of past work on the
value of enrichment schemes for captive species cur-
rently has limited scope because enrichment has neither
explicitly used nor well researched in amphibians (de
Azevedo et al. 2007; Burghardt 2013). The objective of
this paper is to draw attention to this lack of knowledge
Correspondence. Email: "^c.j.michaels44@ gmail.com (Corresponding author).
Amphib. Reptile Conserv. | amphibian-reptile-conservation.org (7)
June 2014 I Volume 8 I Number 1 I ell
Michaels et al.
Table 1. Studies of enrichment in amphibians.
Species
Origin
Type of enrichment
investigated
Findings
Notes
Reference
Xenopus laevis
Unknown
Shelter provision
No effect on growth rate.
Frogs provided with shelter
reluctant to leave it, even
when provided with food.
Small sample size;
unknown origins
and genetics (see
Chum et al. 2013)
Hilken et al. (1995)
Xenopus laevis
Laboratory bred
Shelter provision
Frogs use any shelter
provided, but prefer plastic
tubes to plants, rocks and
wood. Frogs prefer tanks with
shelter to tanks with no shel-
ter. Frogs showed increased
activity and reduced panic in
tanks with shelter.
—
Brown and Nixon
(2004)
Xenopus laevis
Laboratory bred
Shelter provision
Provision of plastic tubes
reduced aggressive en-
counters, wounds and/or
cannibalisation events.
—
Toreilles and
Green (2007)
Xenopus laevis
Laboratory bred
Shelter provision
No effect on growth rates.
Reluctant to leave shelter.
—
Gouchie et al. (2008)
Xenopus laevis
Laboratory bred
Shelter provision
No effect on growth rates or
body condition (fat bodies).
Higher propensity to clump
together without shelter.
—
Archard (2012)
Xenopus laevis
Laboratory bred
tadpoles
1. Surface area size
2. Water depth
3. Aquatic partitioning/
maze
1 . Reduced surface area
increased air-breathing
behavior
2. Shallow water reduced
growth rates and caused
abnormal floating behavior
(tadpoles could not surface
to breath properly)
3. Tadpoles avoided narrower
passages (2 cm) and
preferred wider ones (4
cm)
Enrichments are
not ecologically
relevant to this spe-
cies; this work may
have limited impli-
cations for captive
husbandry
Galich and
Wassersug (2012)
Xenopus laevis
Laboratory bred
females
1 . Shelter provision
2. Conspecific provision
(always with shelter)
1 . Refuge provision reduced
daytime activity and
animals used shelter when
provided
2. Addition of conspecific
further reduced daytime
activity in increased refuge
use. No aggression
observed and refuges were
shared
—
Archard (2013)
Lithobates
catesbeianus
Farmed/wild-caught
Environmental com-
plexity (ramps, perches
and caves)
Improved general welfare
(general aspect and condition
of animals)
High density
laboratory condition
Bang and Mack
(1998)
Lithobates
catesbeianus
Farmed/wild-caught
Shelter provision
Reduction in mortality and
improvement in condition
High density
laboratory condition
Hedge and Saunders
(2002)
Dendrobates
tinctorius
D. azureus
D. auratus
D. leucomelas
Mainly reported
as aggregate data
across species
Zoo bred
1. Feeding enrichment
(control vs. insect
dispenser vs.
broadcast feed/aphid
stem)
2. Enclosure switch
1 . Some effects on behavior
(mainly activity)
2. Effect on activity levels
(enclosure switch lead to
higher activity levels)
Very small sample
sizes. Issues with
experimental
design, includ-
ing few replicates
and unexplained
measures
Hurme et al. (2003)
Oophaga pumilio
Zoo bred
Feeding enrichment
(feeding dish control vs.
feeding dish with leaf
cover to allow insects to
disperse)
Increased foraging duration,
increased duration between
prey capture events and
reduced rapid feeding
—
Campbell-Palmer
et al. (2006)
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June 2014 I Volume 8 I Number 1 I e77
Enrichment for amphibians
Table 1. Studies of enrichment in amphibians (continued).
Species
Origin
Type of enrichment
investigated
Findings
Notes
Reference
Mannophryne
trinitatis
Wild collected as
tadpoles
1 . Shelter provision
2. Substrate type
1 . Strong, positive effect on
growth rates. No effect on
behavior (weak effect on
time spent jumping)
2. Preferred shallow water
Substrate prefer-
ence predicted by
habitat
Walsh and Downie
(2005)
Physalaemus
pustulosus
Wild collected as
spawn
1 . No/weak effect on growth
or behavior
2. Preferred dig-able (sand or
gravel) substrate
Leptodactylus
fuscus
Agalychnis
callidryas
Laboratory bred
juveniles and adults
Shelter provision
Frogs prefer planted to
non-planted enclosures.
This preference increases
when animals are deprived
of plants before choice test.
Froglets reared with plants
grow faster and are in better
condition than those reared
without. Frogs reared with
plants have more diverse and
more abundant cutaneous
bacterial communities.
—
Michaels et al.
(2014b)
Cryptobranchus
alleganiensis
Wild collected as
eggs (head-starting
program)
Pre-release anti-preda-
tor training
Hellbenders were able
to learn to exhibit a fright
response to trout scent after
classical conditioning; control
animals showed no such
improvement.
—
Crane and Mathis
(2010)
and to call for more research in order to better understand
the importance of enrichment for this taxon. We will ex-
plore the meaning of enrichment for amphibians, review
the body of existing research (Table 1), and discuss the
neglect of this field as well as how and why enrichment
may be important as a focus for both amphibian conser-
vation and welfare research activity. Finally, we will sug-
gest a potential structure and goals for future research in
this area (Table 2).
Concepts of enrichment
Enrichment for captive animals has been defined in vari-
ous ways, but in general, is any intervention designed to
improve animal welfare beyond the basic requirements
for survival, usually taking the form of modifications to
enclosures or husbandry protocols. Well known exam-
ples include the provision of bamboo stems filled with
grubs for captive Aye-aye (Daubentonia madagascarien-
sis) (Quinn and Wilson 2004), running wheels for cap-
tive rodents (Hutchinson et al. 2005) and the spraying of
unfamiliar scents on parts of the enclosures for big cats;
e.g., Szokalski et al. 2012 in tigers (Panthera tigris).
Enrichment is often sub-divided into environmental,
behavioral, and social categories. Shepherdson (1998)
defined environmental enrichment as any intervention
that provides “the environmental stimuli necessary for
optimal psychological and physiological well-being.”
This is distinct from behavioral enrichment, which is de-
signed to elicit or allow the expression of specific behav-
Amphib. Reptile Conserv. | amphibian-reptile-conservation.org (9)
iors or behavioral repertoires (Shepherdson 1994). Social
enrichment, the provision of access to other individuals
(usually, but not always, conspecifics) to cater for social
interaction needs (including both environmental and be-
havioral components), has also been identified as impor-
tant for a number of taxa (Berejikian et al. 2001; Lan-
termann 1993; Miranda de la Lama and Mattiello 2010;
Polverino et al. 2012; Saxby et al. 2010; Sloman et al.
2011; reviewed by Hayes et al. 1998 and Young 2003;
see below).
Enrichment can influence behavioral repertoires and
stress levels beyond addressing stereotypical behavior
and physical health problems (reviewed by Young 2003)
and can affect physical brain structure in species as di-
verse as mice {Mus musculus) and crickets {Acheta do-
mestica) (Lomassese et al. 2000; van Praag et al. 2000).
These findings have led to a current view of enrichment,
which recognizes the importance of all three categories
for the psychological as well as the physical welfare of
captive animals (Dawkins 2006; Young 2003).
The three forms of enrichment can be used to improve
conservation success by training animals with the aim of
improving survivorship upon release; e.g., anti-predator
training in the black footed ferret {Mustela nigripes;
Dobson and Lyles 2000). Although some forms of train-
ing may be beneficial, the use of enrichment may result
in conflict between maximizing individual welfare in
captivity and equipping animals destined for release with
the most appropriate survival skills (Caro and Sherman
2013; Harrington et al. 2013), and both objectives should
June 2014 I Volume 8 I Number 1 I ell
Michaels et al.
Table 2. Key areas of species biology knowledge required for effective enrichment research, potential tools for assessing enrich-
ment needs and effects and areas of amphibian captive husbandry for which enrichment may be important.
Key areas of amphibian bioiogy, to be integrated into
enrichment research
Potential measures of welfare and fitness
Potential areas of captive
husbandry for enrichment
research focus
Cognition
Learned and hard- 1
wired behavioral
components |
Catalogue existing issues in 1
captive amphibians and their
husbandry |
Enclosure design
• Size
• Complexity
- Permanent (furniture and
decor)
- Temporal (novel objects,
timed misting)
• Refuges
• Lighting
- Wavelength
- Photoperiod
- Intensity
Perception of
environment
1
Behavior and behavioral
assays
1
Behavior
1
Natural behav-
ioral repertoires and i
activity levels of 1
species
1
Foraging success
1
Environmental parameters
• Gradients
• Fluctuation (seasonal
and diel)
Foraging strategies '
and dietary compo- =
sition 1 >
1 O
1 <r>
15
Growth and development
1 ^
1
Reproductive ^
behavior g,
• Breeding "g
strategies ^
• Mate choice . o.
• Competition for | -g
mates/breeding S
sites 1 Q.
s-2-
1 ns
<D
1 1
Body condition c
1 ^
1 1
Q.
O
1 ^
Threat stimuli
• Predation
• Competition
• Environmental stressors
(e.g., drying ponds)
^ ■(«
Migration and home i ^
ranges ' "5
Hormones i
• Stress 1 w
• Reproductive ' «
O)
1 ^
Encouraging specific
behavioral responses
1 ^
1
c
1 ^
ns
Antipredator
behavior . -2
1
Micro- and macro-biotas o
associated with animals . =
1 CD
• Beneficial communities 1 ^
(mainly skin and gut) o
• Parasite and pathogen 1
loads
1
Nutrition and food presentation
• Nutritional content
• Temporal variation
• Variation in food types
(different species of prey
animal or algae)
• Total abundance
1
“Personality” vs be- 1
havioral plasticity 1
1
Pathologies '
• Behavioral
• Physical (disease, 1
malformation and 1
pathogen susceptibility)
1
Interactions
Intra- and inter-
specific 1
1
Reproductive success |
1
Social enrichment
• Presence of conspecifics
and non-conspecifics
• Stability of social groups
• Territory creation and
maintenance
• Mate choice
• Human habituation
As predators, prey
and competitors .
Genetics and
evolution
1
Heritability of traits
1
Survivorship 1
1
Potential for
selection |
be considered for conservation breeding populations.
The ferrets trained for release, for example, although
not physically harmed, would have been psychologi-
cally distressed by being pursued by muzzled dogs as is
a prerequisite of successful aversive training. This topic
will continue to be controversial, as it is impossible to
objectively resolve the relative importance of individual
welfare and the persistence of a species as a whole, or
whether the compromise of one is worth the assurance
of the other. However, it is important to consider the in-
dividual welfare gains of such training post release. Pre-
release anti-predator training may compromise welfare
of animals in captivity, but may result in a larger welfare
gain, when animals avoid predators in the wild.
Burghardt (1996) suggested that the term “controlled
deprivation” might be more appropriate than “enrich-
ment.” This term acknowledges that it is impossible to
provide in captivity the level of stimulation gained by
animals in the wild, but rather management strategies
should seek to strategically provide stimulation in such a
way as to control the effects of general deprivation. The
term “enrichment” may suggest a positive increase in
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June 2014 I Volume 8 I Number 1 I e77
Enrichment for amphibians
stimulation due to management strategies, when in faet
it is not. While “controlled deprivation” is perhaps more
honest, the vast majority of work continues to use the
term “enrichment.” We will therefore continue to do so,
but with the caveat that such strategies enrich the life of
captive animals compared with captive life devoid of any
stimulation, rather than compared with what they might
receive in the wild.
The conceptual framework of enrichment has largely
focused on birds and mammals, and it may be problemat-
ic to apply it consistently when assessing enrichment for
amphibians, particularly because the distinction between
environmental and behavioral enrichment is blurred.
Amphibian behaviors are often linked to specific physio-
logical functions, such as basking, hunting or burrowing,
or to reproduction, so we will not differentiate between
these two enrichment types. Additionally, the highly spe-
cific environmental requirements of captive amphibians
mean that many aspects of amphibian husbandry, such
as UVB provision (Antwis and Browne 2009) and nutri-
tion (e.g., Antwis et al. 2014; Li et al. 2009; Ogilvy et al.
20 1 2a, b), impact both basic requirements and enrichment
as described by Shepherdson (1998). The relative lack of
empirical work in this field further hinders differentia-
tion between different enrichment categories. We opt to
exclude aspects of husbandry that offer benefits only to
“physiological well-being,” in order to allow a focus on
true enrichment that transcends basic husbandry. Within
this category, there is a distinction between enrichment
solutions that simply provide animals with things that
they have evolved to psychologically rely upon and those
that offer specific learning opportunities. The provision
of shelter may fall into the former category, for example,
while training amphibians to avoid predators may be in-
cluded in the latter. Both may be important to consider,
although learning-oriented enrichment may be of greater
significance to animals intended for release.
The neglect of amphibian enrichment research
Within the conservation and animal welfare literature
there is a lack of research on amphibians and reptiles
compared with the other tetrapod vertebrates (de Azave-
do et al. 2007; Bonnet et al. 2002; Griffiths and Pavajeau
2008; Griffiths and Dos Santos 2012) and the body of
published work in the area of enrichment for amphibians
is limited (Table 1).
Amphibians, like all ectotherms, have historically
been perceived as animals that cannot suffer, or do not
feel pain, at least to the same degree as mammals and
birds (Gross 2003). This bias has meant that the use of
anaesthetics and analgesics during amphibian veterinary
care and surgical procedures in the laboratory and field
is relatively recent (Machin 1999). Although arguments
have been made to suggest that amphibians (and fish) do
not exhibit consciousness or emotion, while the amni-
otes do to varying degrees (reviewed by Cabanac, et al.
Amphib. Reptile Conserv. | amphibian-reptile-conservation.org (11)
2009), this is by no means conclusive. The identification
of pain pathways shared between amphibians and other
amniotes (Stevens 2004) suggests an ability to experi-
ence pain, even if in a different and more restricted sense
than in amniote taxa. This argument notwithstanding,
the capacity to suffer in the presence of pain does not
influence the importance of enrichment for conservation
purposes.
Additionally, amphibian behavioral motivations, the
reasons animals exhibit a particular behavior, are more
difficult for humans to intuit than those of mammals and,
to a lesser extent, birds, both of which may engage in be-
haviors more easily recognized by humans. Along with
a lack of available, amphibian-specific measures of wel-
fare, the difficulty in instinctively understanding amphib-
ian behavioral motivations may have reduced interest in
enrichment for this group as there may be fewer easily
noticed welfare problems. Furthermore, the reliance of
many amphibian species on highly specific environmen-
tal conditions often necessitates more complex and often
“naturalistic” environments than would be required to
maintain and breed mammals or birds, or even many rep-
tiles. Consequently obvious symptoms of extreme depri-
vation may be less apparent, unlike in other taxa that may
survive and reproduce in confined and bare enclosures,
the more complex environmental requirements of some
amphibians may be more difficult to disentangle from
their basic husbandry. The rapidity with which many am-
phibians physiologically succumb to poor environmental
conditions (Wright and Whitaker 2001) may not allow
the development of any potential behavioral abnormali-
ties before an animal dies. Moreover, the reduced activity
in many contexts and lower metabolic capacity of many
amphibians may reduce or mask the appearance of ac-
tive behavioral stereotypes in some taxa. Additionally,
increased stress hormone levels have been associated
with a downregulation of behaviors, including reproduc-
tion (Moore and Miller 1984; Moore and Zoeller 1985;
Chrousos 1997; Moore and Jessop 2003) and foraging
(Crespi and Denver 2005; Carr et al. 2002), in some am-
phibians and so the effects of poor enrichment may, in
some cases, manifest as absences of normal behavior in-
stead deviant or new behaviors.
The relatively innate, “hard-wired” behavior of am-
phibians is often used to support the idea that enrich-
ment, and consequently research investigating it, is not
an important consideration, particularly in ex situ conser-
vation (Bloxam and Tonge 1995; Griffiths and Pavajeau
2008). Some forms of enrichment involve learning (e.g.,
antipredator behavior learning; Dobson and Lyles 2000),
whereas others may simply allow the manifestation of
behaviors without a learning component. Although am-
phibians may not rely on captive conditions to develop
normal behavioral repertoires as mammals or birds, their
behaviors can be complex (reviewed by Burghardt 2013)
and the role of learning is more important (reviewed by
Bee et al. 2012; Wells 2007) than was previously thought.
June 2014 I Volume 8 I Number 1 I ell
Michaels et al.
Research on enrichment in amphibians,
reptiies and fish
Measuring the impact of enrichment on amphibians
Objective measures of amphibian welfare have not been
well developed or validated, beyond major issues such as
cannibalism and bite trauma (Toreilles and Green 2007).
Stereotypical behaviors in amphibians are poorly defined
or understood (there is no mention of behavioral prob-
lems in Wright and Whitaker’s (2001) otherwise com-
prehensive amphibian medicine and captive husbandry
volume), and are usually only recognized in the form of
gross trauma. It is likely that abnormal and stereotypical
behaviors frequently used to assess welfare in mammals
and birds may not be applicable to amphibians. More-
over, a number of commonly used measures are subject
to a priori assumptions about their interpretation and,
although they may seem reasonable, good rationales for
the use and interpretation of characters as measures of
welfare are rarely given. Activity levels have been used
(Archard 2013; Campbell-Palmer et al. 2006; Hurme et
al. 2003), but the conclusion that particular effects (e.g.,
increased foraging time or reduced daytime activity)
translate to improved welfare remain largely untested as-
sumptions. Similarly, authors generally interpret faster
growth rates and larger fat bodies as indicators of better
welfare, as well as being indicative of the production of
more robust individuals. Dawkins’ (1983; 1990) “con-
sumer demand” methodology to assess animal needs has
not been applied to amphibians, although choice cham-
bers have been used to assess preferences (Michaels et al.
2014b; Walsh and Downie 2005). In reptiles, trade-offs
between palatable food and cold temperatures have been
used to assess the “consumer value” of a food reward to
green iguana (Iguana iguana; Balasko and Cabanc 1998)
and this methodology could be applied to amphibians.
Corticosteroid or “stress” hormone levels have been
used to assess welfare in amphibians (Coddington and
Cree 1995; Narayan et al. 2010, 2011a, b; Narayan and
Hero 2011; Paolucci et al. 1990; Zerani et al. 1991), but
beyond easily interpreted contexts such as capture, trans-
port, and toe clipping, they can be problematic. In par-
ticular, a lack of baseline data across different contexts
for most species makes interpretation, in terms of wel-
fare, of isolated samples difficult. “Stress” is best viewed
in its evolutionary, physiological, genetic, ecological,
and behavioral contexts (Boonstra 2013) and increased
levels are associated with and necessary for normal be-
haviors including reproduction (Moore and Jessop 2003;
Narayan et al. 2010), immune responses (Rollins-Smith
2001), and adaptive plasticity (Denver 1997). “Stress”
and “distress” are very different states, with only the lat-
ter having negative impacts on animal fitness and wel-
fare, and these must be considered separately (Linklater
and Gedir 2011). However, non-endocrine, unambiguous
measures of welfare must be developed in order to prop-
Amphib. Reptile Conserv. | amphibian-reptile-conservation.org (12)
erly distinguish between stress, which may be normally
physiologically elevated in certain contexts, and distress
in amphibians. Measurements of suites, instead of iso-
lated, characters (e.g., Michaels et al. 2014b) will help
to build a more easily interpreted picture of the effects
of enrichment. Assessment of symbiotic or mutualistic
bacterial communities on the physiologically active skin
of amphibians may provide a new measure of welfare.
These communities are sensitive to facets of enclosure
design that can also be shown to impact other “tradi-
tional” measures of welfare and fitness including growth
rates, body condition, behavior, and reproductive output
(Antwis et al. 2014; Michaels et al. 2014b) as well as
corticosteroid levels following challenges (R. Antwis,
unpublished data). Although these communities do not
allow distinction between stress and distress, they pro-
vide an additional line of enquiry in this area. Given the
important impact of microbial communities on disease
resistance (Bletz et al. 2013), this field can provide strong
links between enrichment and the likelihood of reintro-
duction success.
Importantly, any evidence must be interpreted in the
context of the focal species (Michaels et al. 2014a). In-
creased activity levels, for example, are more likely to
be beneficial in actively hunting species than in ambush
predators that do not typically engage in extended loco-
motion. Comparison between wild and captive conspe-
cifics may provide guide “targets” for developmental and
physiological measures, such as body condition, as well
as a means to establish natural behavioral repertoires.
Existing enrichment research in amphibians
We identified 14 primary research articles on amphibian
enrichment, summarized in Table 1, all but one (Crane
and Mathis’ (2010) hellbender training study; see below)
of which were concerned primarily with improving indi-
vidual welfare of captive animals, as opposed to improv-
ing breeding or release success. In some cases, the impact
of enrichment has not been investigated beyond a subjec-
tive assessment of “appreciation” by people and practi-
cality (e.g., Hanley 1993; Kirkland and Poole 2002) and
such work has not been included in this count. Burghardt
(2013) reviewed evidence for the effects of enrichment
in both reptiles and amphibians, but did not include some
of the studies discussed here. Furthermore, the focus of
his review was on cognition and its implications for the
understanding of enrichment for reptiles and amphibians,
as well as a consideration of evidence for consciousness,
play, and emotion in these groups. There was no discus-
sion of pre-release training or the role of enrichment in
conservation for amphibians.
Shelter provision is the most investigated form of en-
richment for amphibians, including the common model
organism Xenopus laevis (reviewed by Chum et al. 2013;
Tinsley 2010; see Table 1), and in five other species
(Physalaemus pustulosus, Leptodactylus fuscus, Man-
June 2014 I Volume 8 I Number 1 I ell
Enrichment for amphibians
nophryne trinitatis, Agalychnis callidryas, and Litho-
bates catesbeianus; Table 1). Although shelter provision
undoubtedly has physiologieal benefits for amphibians
(Miehaels et al. 2014b; Walsh and Downie 2005), behav-
ioral tests (see Table 1) have suggested a psychologieal
element to the effects of shelter provision, implying that
it falls within our definition of enrichment for amphib-
ians. However, more comprehensive investigations of
this are warranted.
The conclusions of this literature are somewhat mixed,
particularly for Xenopus but in general support the im-
portance of shelter provision for frogs studied (Archard
2013; Chum et al. 2013; Bang and Mack 1998; Hedge
and Saunders 2002; Michaels et al. 2014b; Tinsley 2010;
Walsh and Downie 2005; Table 1). In non-Xenopus spe-
cies, multiple measures of welfare and fitness all show
improvements in the presence of enrichment. In Xeno-
pus, changes in behavior do not seem to be reflected in
growth rates or body condition, nor are these negatively
affected by enrichment. These differences between taxa
in response to the same type of enrichment (shelter provi-
sion) are indicative of the limited degree to which find-
ings from one species can be applied to others, and the
need for the development of species-specific measures of
welfare. They also highlight the importance of measuring
a number of variables in response to enrichment.
Two studies investigate enrichment through environ-
mental complexity beyond shelter provision. Bang and
Mack (1998) showed that increased general environ-
mental complexity in the form of ramps, perches, and
caves positively affected the welfare of captive bull-
frogs {Lithobates catesbeianus’. Table 1), although it is
unclear if this extended beyond the effects of shelter
alone (Hedge and Saunders 2002). Calich and Wasser-
sug (2012) found impacts of water depth, surface-area
size and aquatic partitioning on the behavior of X. laevis
tadpoles, but the enclosure modifications were not eco-
logically relevant to this open-water species (Tinsley and
Kobel 1996) and the findings are perhaps of limited use
in developing husbandry protocols.
Food-delivery enrichment affects behavior and activ-
ity levels in dendrobatid frogs (Campbell-Palmer et al.
2006; Hurme et al. 2003), whereas introduction of frogs
to novel environments also increased activity levels
(Hurme et al. 2003). Archard (2013) investigated the ef-
fect of social enrichment, through the provision of con-
specifics, in an enclosure containing a refuge, as well as
the effect of shelter per se (see above). The author found
that X laevis exhibited reduced da)hime activity, beyond
the reduction seen when refiigia are provided, when con-
specifics are present in tanks with shelter. This result was
interpreted as an improvement in welfare, but such and
interpretation may be viewed as ambiguous, particularly
in a species known to show a degree of territoriality in
the wild (Tinsley and Kobel 1996).
One study has investigated the use of enrichment to
train hellbenders {Cryptobranchus alleganiensis) for
Amphib. Reptile Conserv. | amphibian-reptile-conservation.org (13)
release into the wild. Crane and Mathis (2010) used a
combination of trout-scented water and conspecific dis-
tress secretions to train hellbender larvae in head-starting
programs to avoid predation by predatory trout. This
pre-release training may be classed as a form of enrich-
ment for these salamanders, encouraging them to express
normal anti-predator behavior, but manipulating this to
improve future survival in the face of invasive alien pred-
ators. Several classes of amphibian behavior have now
been shown to include learned components, including
predator avoidance (Crane and Mathis 2010 in Crypto-
branchus alleganiensis’, Epp and Gabor 2008 in Eurycea
nana’, Ferrari and Chivers 2008 in several species of an-
uran larvae), territoriality (Dawson and Ryan 2009; 2012
in Physalaemus pustulosus), foraging (Sontag et al. 2006
in anuran larvae) and other aspects of social behavior
(Bee et al. 2012; Wells 2007). Moreover, complexity and
cognition, whereby behavioral processes exceed simple
responses to stimuli, have been detected in a range of
amphibian behaviors, including spatial learning and
homing (Brattstrom, 1990; Shoop 1965) and individual
recognition (Gauthier and Miaud 2003). Amphibians are
also capable of visual discrimination learning, identify-
ing objects based on visual characteristics, (Jenkin and
Laberge 2010) and even rudimentary quantity learning,
showing the capacity to compare quantities, (Krusche et
al. 2010; Uller et al. 2003). Although these findings have
implications for all areas of enrichment for amphibians,
they suggest that enrichment in captivity might have par-
ticular applications in pre-release training. However, ap-
plying this increased knowledge of amphibian learning
and behavioral complexity to enrichment has not been
empirically tested (apart from the aforementioned hell-
bender study). Furthermore, in the context of predation
the ethics of any compromise between welfare and long-
term reintroduction success must be carefully considered
(Caro and Sherman 2013; Harrington et al. 2013).
Some of the research investigating enrichment for
amphibians is problematic in terms of sample size and
experimental design. Hurme et al. (2003) could not de-
tect significance in some effects due to extremely limited
sample size. Walsh and Downie (2005) used a sample
size suitable for statistical analysis, but in their cover
provision experiments, fossorial or semi-fossorial anuran
species (Leptodactylus fuscus and Physalaemus pustu-
losus) were provided with a soft substrate in enclosures
both with and without cover. As the authors admit, it is
likely that the effects of cover provision in these species
were weaker in comparison with the non-fossorial third
study species {Mannophryne trinitatis) due to this soft
substrate acting as “cover” for the frogs, which could
simply burrow in order to hide.
Enrichment research in amphibians is subject to
strong taxonomic bias in addition to bias towards shelter
provision. Half of the articles (seven of 15, Table 1) used
X. laevis as a study species, while of the other species
used, six of eleven were dendrobatoid frogs and only one
June 2014 I Volume 8 I Number 1 I e77
Michaels et al.
caudate was represented (Table 1). To our best knowl-
edge, there has been, to date, no explieit researeh on en-
riehment for caeeilians (Gymnophiona). However, one
biomeehanics study (Ducey et al. 1993) may be relevant
to caecilian enrichment, as it demonstrates that caeeilians
of four fossorial species {Ichthyophis kohtaoensis, Der-
mophis mexicanus, Gymnopis syntrema, and Schistome-
topum thomense) preferred and were most capable of
digging in uneompacted soil, and that they use existing
burrows rather than eonstrueting new ones if given the
ehoice. This concurs with field studies, which have gen-
erally found terrestrial caeeilians in looser, more friable
soil and leaf-litter in established burrow systems (Kupfer
et al. 2005; Malonza and Measey 2005; Measey 2004;
Oomen et al. 2000; Habidata.eo.uk).
Comparison with fish and reptile literature
For amphibians, given the narrowness of enrichment
types investigated and the limited range of focal species
(both taxonomically and ecologieally), it is diffieult to
extrapolate eurrent evidenee to other amphibians and
to other enrichment types. In order to prediet the im-
portance of enriehment for amphibians, therefore, we
examined evidence from the two vertebrate taxa most
similar to amphibians: reptiles and fish. Despite the fact
that mammals and birds are better studied (de Azevedo et
al. 2007), reptiles and fish are generally more similar to
amphibians in neurologieal eomplexity, cognitive ability,
physiology, and ecology. The literature for fish is much
larger than for amphibians and that for reptiles is both
larger and includes a wider range of enrichment types (de
Azevedo et al. 2007). We do not suggest that these groups
are identieal in their needs, but until advances in amphib-
ian enriehment researeh are forthcoming, inference from
these taxa may be important to consider. Furthermore,
methodologies used to assess enriehment in reptiles and
fish may easily transfer to the study of amphibians.
Researeh on fish has focused largely on the eommer-
eial improvement of fisheries, the improvement of fit-
ness in animals intended for release to the wild, and to
a lesser degree on the welfare of fish speeies eommonly
used in biomedical research. Enrichment through envi-
ronmental eomplexity generally improves eognitive and
learning ability in fish (Brown and Braithwaite 2005 in
Brachyraphis episcope; reviewed by Strand et al. 2010),
reduee stress and stress-related behavior and metabolie
activity (Batzina and Karakatsouli 2012 in Sparus au-
ratus; Finstad et al. 2007 in Salmo salar; Millidine et
al. 2006 in S. salar; Zimmerman et al. 2012 in Gadus
morhua), increases behavioral plasticity (Berejikian et
al. 2001 in Onychorhyncus mykiss), increases territory
holding power (Nijmen and Heuts 2000 in a variety of
speeies) and improves foraging, risk assessment, and
predator-avoidance behavior (Braithwaite and Salvanes
2005 in G. morhua; Brown et al. 1998 in O. mykiss;
Brown et al. 2003 in S. salar; Lee and Berejikian 2008
Amphib. Reptile Conserv. | amphibian-reptile-conservation.org (14)
in O. mykiss; Roberts et al. 2011 in S. salar). Moberg et
al. (2011) found inereased timidity of G. morhua reared
in enriched hatchery conditions once exposed to a novel
arena, possibly due to less developed coping strategies in
animals reared with shelter. This body of evidenee should
stimulate interest in similar phenomena linked to envi-
ronmental eomplexity in amphibians, whieh could have
important implieations for the success of release or re-
introduetion projects. It seems that innate, “hard- wired”
fish behavior ean be enhaneed and honed by enrichment
in the form of exposure to simulated predator disturbanee
(Berejikian et al. 2003 in O. tshawytscha) or by soeial
learning (Vilhunen et al. 2005 in Salvelinus alpinus; re-
viewed by Brown and Laland 2001). The similarity to the
limited literature on eomparable phenomena in amphib-
ians (Crane and Mathis 2010; Epp and Gabor 2008; Fer-
rari and Chivers 2008; Sontag et al. 2006) suggests that
there is much to learn about the application of amphibian
learning to captive husbandry and pre-release training.
Enrichment in fish farms also improves growth rates,
similar to the effects of shelter provision in amphibian
speeies (Archard 2013; Chum et al. 2013; Tinsley 2010;
Walsh and Downie 2005), inereases potential stocking
densities and reduces aggression (Batzina and Karakat-
souli 2012; Finstad et al. 2007), as does enriehment in
amphibians (X laevis; Toreilles and Green 2007). The
impacts of enrichment may be trans-generational; Evans
et al. (2014) found that adult farmed salmon (S. salar) in
enclosures enriched by exposure to wild conditions while
in captivity produced offspring with a two-fold increase
in survivorship eompared with fish maintained under
standard farm conditions. Given the normal use of pre-
release training only in the individuals to be exposed to
predation (Crane and Mathis 2010), it may be important
to investigate trans-generational effects of enrichment in
amphibians.
A few studies have focused on individual welfare in
laboratory and aquarium fish speeies, but as for amphib-
ians these have mainly investigated cover provision. This
work has, surprisingly, found little benefit to providing
enriehment in laboratory aquaria, in the form of eover/
environmental eomplexity, with fish often showing no
differences in growth rates or stress-hormone levels
(Brydges and Braithwaite 2009 in Gasterosteus aculea-
tus; Wilkes et al. 2012 in Danio rerio), although these are
perhaps not eomprehensive measures of welfare. Kistler
et al. (2011), however, found a preferenee for structured,
rather than barren, environments in both D. rerio and
the barb Puntius oligolepis. These eontradietory results
may partly be due to the highly constrained nature of en-
riehment solutions within strictly controlled laboratory
conditions. The glass rods provided as enrichment for
zebrafish by Wilkes et al. (2012) may not have been suf-
ficient to generate a beneficial effect, whereas the plants
and hides provided in the preference study of Kistler et
al. (2011) may have been complex enough to generate
a detectable behavioral response in the same species.
June 2014 I Volume 8 I Number 1 I ell
Enrichment for amphibians
Furthermore, as neither study analyzed both behavioral
and developmental/endoerine data, it is possible that any
improvement to welfare did not translate to all measures.
Saxby et al. (2010) and Sloman et al. (2011) found
evidence for welfare and behavioral benefits of social en-
richment in terms of both increased group size and mixed
species assemblages in a variety of fish species common-
ly kept in home aquaria. Similarly, schooling and mixed
species assemblages are common in anuran tadpoles in
the wild and may have implications for learning (Ferrari
and drivers 2008; Sontag et al. 2006); the application of
this for conservation breeding may be important to con-
sider.
Reptiles have been better studied than amphibians in
terms of enrichment research (de Azevedo 2007; Hayes
et al. 1998) and attempts have been made in reptiles to
identify and define stereotypical behavior and to sug-
gest aetiologies (Bels 1989; Hayes et al. 1998; Warwick
1990). This literature is more focused on individual wel-
fare of captive animals than is the fish literature and has
involved zoo animals, as opposed to farms. Small sample
sizes and anecdotal reports are a common problem in
the reptile enrichment literature and much of it includes
reasoned suggestions for enrichment, rather than empiri-
cal evidence of its efficacy (Burghardt 2013; Hayes et al.
1998). For this reason, enrichment solutions are, in gen-
eral, more suitable for short-term use by a small group of
animals, in contrast to the types of larger-scale enrich-
ment often investigated in fish.
Captive conditions alter and reverse wild patterns of
antipredator behavior of reptiles (Hennig and Dunlap
1978; Hennig 1979, both in Anolis carolinensis) and
strike-induced chemosensory searching (“scent-trailing;”
Marmie et al. 1990 in Crotalus enyo). The provision of
a complex environment in captivity improves cognitive
behavior (Almli and Burghardt 2006 in Elaphe obsoleta)
and reduces stress hormone levels and stress-related es-
cape behavior (Case et al. 2005 in Terrapene Carolina).
Blue-tongue skinks {Tiliqua scincoides) show alterations
to activity patterns and exhibit reduced weight gain and
obesity when provided with larger enclosures and the op-
portunity to hunt for insect prey (Phillips et al. 2011).
Complex environments are also actively sought out by
reptiles (Case et al. 2005 in T. Carolina), while individu-
als of cryptic species may also seek out and prefer ap-
propriately colored refiigia (Garrett and Smith 1994 in
Morelia viridis), as do wild amphibians (Pacific tree-
frogs, Pseudacris regilla; Morey 1990). Furthermore, al-
though sometimes controversial (Burghardt 2005), some
reptiles have been reported to engage in divertive, play
behavior when provided with novel objects (Burghardt
et al. 1966 and Burghardt 2005 in Trionyx triunguis; Hill
1946, Murphy 2002 and Burghardt 2005 in Varanus ko-
modoensis; Lazell and Spitzer 1977 m Alligator mis sis -
sippiensis). Animals have also exhibited a reduction in
self-mutilation (Burghardt et al. 1996) and engaged in
normal behavioral repertoires instead of apathy or ste-
Amphib. Reptile Conserv. | amphibian-reptile-conservation.org (15)
reotyping when provided with such enrichment (Ther-
rien et al. 2007 in Caretta caretta and Chelonia mydas).
Also, monitors {Varanus albigularis and V. rudicollis)
and anoles {Anolis evermanni) were capable of rapidly
learning to solve cognitively demanding tasks (Gaalema
2011; Leal and Powell 2012; Manrod et al. 2008).
In contrast, Marmie et al. (1990) found no differences
between groups of rattlesnakes {Crotalus enyo) raised in
large or small enclosures, and wild conspecifics, in their
ability to explore novel environments. Likewise, Rosier
and Langkilde (2011) found no differences in Scelopo-
rus undulatus behavior, stress hormone levels, survivor-
ship and growth when a complex environment (climbing
space) was provided. However, the small size and rela-
tive simplicity of the enclosures utilized in these cases
may not have provided the degree of complexity required
to provide effective enrichment for these animals: there
has been some discussion of the validity of experimental
design (see Burghardt 2013 for a summary of this ex-
change).
Finally, a few studies in reptiles examined the rela-
tionship between enrichment and survival in reintro-
duced animals, with encouraging results. Cook et al.
(1978) reported the use of enrichment in the form of
pre-release desert survival training of captive desert tor-
toises {Gopherus agassizii) in California and suggested
that this approach improved survival from 0% in earlier
release trials to 70% in trained tortoises. Although pre-
release enrichment and training may have improved re-
lease success, rehabilitation centers also treated tortoises
for a host of diseases that do not seem to have been ad-
dressed in earlier reintroduction attempts (the documen-
tation is unclear), so the true impact of training is difficult
to ascertain. Price-Rees et al. (2013) reported a similar
training effort in blue-tongue skinks {Tiliqua scincoides
intermedia), where aversive training was used to pre-
vent lizards from eating lethally toxic cane toads {Rhi-
nella marina), with large improvements in survivorship
compared with control skinks. These findings reinforce
the need for further investigation into the role of enrich-
ment in pre-release training for amphibians. They also
highlight the potential for such slightly aversive training
to significantly improve both the welfare of individuals
released into the wild and the success of conservation
initiatives.
What impacts might enrichment have for cap-
tive amphibians?
Impacts on welfare
Enrichment has been demonstrated to reduce mortality
and injury in some amphibians and to improve growth
rates and body condition in others (Table 1). Further-
more, the majority of amphibian diseases found in cap-
tive populations and regularly treated by specialist vet-
erinarians are related to improper husbandry (Wright and
June 2014 I Volume 8 I Number 1 I ell
Michaels et al.
Whitaker 2001). Obesity, metabolie bone disease (MBD)
and related nutritional disorders are eommon problems in
eaptivity (Gagliardo et al. 2008; Lee et al. 2006; Wright
and Whitaker 2001). Enrichment designed to increase the
effort required to forage for food (alongside a balanced
diet; Li et al. 2009) increased activity levels (Campbell-
Palmer et al. 2006) and, for actively foraging species (see
below), should re-balance energy budgets while allowing
animals to satiate their hunger, as has been demonstrat-
ed in skinks (Phillips et al. 2011) and cats (Clarke et al.
2005). Likewise, enrichment to encourage basking be-
havior in appropriate species (e.g., Pelophylax lessonae,
which spend considerable portions of the day in the wild
basking in sunlight; Michaels and Preziosi 2013), along-
side the provision of Ultraviolet B radiation in suitable
doses and gradients, is likely to be important in facilitat-
ing calcium uptake from the gut in many species, thus
avoiding clinical and subclinical Metabolic Bone Dis-
ease (MBD) (Antwis and Browne 2009; Verschooren et
al. 2011). Alongside basic facilitation via perches and
basking sites, the provision of shelter and environmental
complexity may alleviate perceived predation pressure
and encourage basking behavior.
Beyond effects on the health and physical welfare
of captive amphibians, enrichment may also have im-
plications for psychological welfare. Enrichment may
improve the cognitive engagement and capacity of am-
phibians, as has been shown in both reptiles and fish, as
well as allowing animals to avoid perceived predation
pressure (Michaels et al. 2014b). Eurther work is needed,
however, to address these issues and to establish how en-
richment may influence psychological well-being.
Implications for conservation
Enrichment may improve the success of reintroduction
and head-starting programs in amphibian conservation.
Evidence from amphibians, reptiles and fish strongly
suggests that enrichment can influence a suite of char-
acteristics, from growth rates to anti-predator behavior,
which may influence the success of reintroductions.
Furthermore, the potential for trans-generational effects
warrants investigation in captivity. The provision of en-
richment may influence survival and reproduction and
consequently the genetic changes that occur over mul-
tiple generations, generating animals adapted to a captive
environment (Frankham 2008). Genetic adaptation to
captivity, or domestication, occurs due to differences be-
tween the wild and captive environment via genetic drift,
founder effects, the unintentional selection for animals
suited to the captive environment rather than the wild
habitat into which they will eventually be released, or
a combination these forces (Frankham 2008). Evidence
for this phenomenon has been found in a wide range of
breeding programs (reviewed Witzenberger and Hoch-
kirch 2011) may be evident in a single generation (Chris-
tie et al. 2012). Amphibians are no exception, and adap-
Amphib. Reptile Conserv. | amphibian-reptile-conservation.org (16)
tation to captivity has been detected in this group. For
example, lack of exposure to predator cues and predation
pressure resulted in loss of anti-predator behavior in the
tadpoles of Alytes mulletensis after 8-12 generations in
captivity in association with a reduction in genetic diver-
sity (Kraaijeveld-Smit et al. 2006). Although many cap-
tive breeding programs run studbooks to preserve genetic
diversity and avoid genetic adaptation to captivity, these
may fail due to unrealistic model assumptions (Witzen-
berger and Hochkirch 2011). In amphibian studbooks,
tadpoles do not tend to be included as individuals and
so populations may suffer non-random mortality before
allele frequency changes can be prevented. The high
fecundity of many amphibians means that most larvae
cannot be raised to adulthood and necessary culls often
remove tadpoles or metamorphs perceived to be weaker
or smaller (C. Michaels, per. observ.). The use of enrich-
ment to sort behaviorally fit and less fit animals, for ex-
ample in response to predator cues, may be a more valid
basis for culls than, for example, body size, although this
idea is inevitably a source of ethical controversy (Caro
and Sherman 2013; Harrington et al. 2013). Appropri-
ately applied enrichment may also prevent more domes-
ticated animals from gaining reproductive advantages in
captivity. For example, animals that are unable to hunt
effectively, but are capable of producing large numbers
of young and readily reproduce in captivity may contrib-
ute disproportionately to programs unless animals are
forced to forage more naturally for prey. Similarly, the
use of enrichment may allow less domesticated animals
to thrive in captivity, where they may be lost from breed-
ing programs if housed without appropriate stimulation.
Finally, non-genetic inherited traits (“maternal” or
“parentaf’ effects) are becoming increasingly recognized
as important in evolutionary terms. The genetic or envi-
ronmental background of parents can influence offspring
phenotype regardless of the genetic correlation between
parents and offspring (Marshall and Uller 2007; Mous-
seau and Fox 1998). Epigenetic effects may improve
or reduce offspring fitness, depending on the system
and circumstances and can influence a wide range of
characters in most plant and animal taxa (Franklin and
Mansuy 2010; Marshall and Uller 2007; Mousseau and
Dingle 1991; Mousseau and Fox 1998; Roach and Wulff
1987). Epigenetic effects have been reported in a num-
ber of amphibian taxa (including Kaplan 1987; Kaplan
and Philips 2006; Pakkasmaa et al. 2003; Parichy and
Kaplan 1992; Rasanen et al. 2003) and are of increasing
importance in the consideration of animal behavior and
welfare (reviewed Jensen 2014). They may be linked to
the degree of enrichment in the captive environment, al-
though this has not been studied in amphibians. McCor-
mick (2006), for example, found that crowding in a num-
ber of marine fish species resulted in decreased fitness,
regardless of their genotype, of offspring, independent of
genotype, even when offspring were raised under identi-
cal, spacious conditions. Similarly, Evans et al. (2014)
June 2014 I Volume 8 I Number 1 I ell
Enrichment for amphibians
demonstrated trans-generational effeets of enriehment in
salmon bred for conservation, such that enriching paren-
tal enclosures improved post-release survivorship in off-
spring. Enrichment for captive amphibians therefore has
the potential to influence the fitness of future generations
through both epigenetic and genetic effects. Importantly,
the phenotype (and therefore chance of survival in the
wild) of an individual is determined by the interaction
between genes and the environment (including both di-
rect and epigenetic/parental components), both of which
can be partially determined by the enrichment strategies
employed in captivity.
As these effects cannot be controlled through stud-
books, it may be of great importance to provide a de-
gree of enrichment that does not encourage epigenetic
changes in captive amphibians.
Future directions for research
Being at the early stage of enrichment research in am-
phibians means that little is known of its impact on wel-
fare and fitness or which types of enrichment may be im-
portant. Amphibian captive welfare and methods suitable
for measuring it are poorly understood or underdevel-
oped in comparison with other taxa. Given the urgency
to provide answers for ex situ conservation projects (Gas-
con et al. 2005) it is important to develop enrichment re-
search goals and priorities. Table 2 outlines a potential
structure for enrichment research in amphibians. Most
areas of amphibian husbandry are strongly constrained
by the natural history of the species in question (Mi-
chaels et al. 2014a) and needs and responses to captive
stimuli vary greatly among taxa and sometimes between
populations (e.g., Tidwell et al. 2013). A more thorough
understanding of the biology of focal species can aid in
the design of meaningful enrichment and experiments.
Consequently, we recommend that researchers first de-
velop a good understanding of the biology of focal spe-
cies before attempting to develop and evaluate enrich-
ment activities. Based on this knowledge, experimental
methods and measures of welfare can be developed and
areas both already identified as important in amphibians,
and those highlighted by work in fish and reptiles, can be
investigated. It is important to develop objective mea-
sures of welfare, including identification of stereotypical
or abnormal behaviors in captive amphibians. Ideally, re-
searchers should aim to use as many different measures
of welfare and fitness as possible in order to develop the
best possible picture of the effects of enrichment. Com-
parisons between wild and captive conspecifics may also
help with this process, particularly where enrichment is
intended to improve the suitability of animals for release.
Objective measures of welfare may also aid in address-
ing conflicts between training required for improved re-
introduction success and ensuring that animals are not
distressed while in human care.
Collaboration between research institutions, which
have the experimental expertise to carry out meaning-
ful research, and zoological collections, which have
access to animals and species-specific knowledge may
expedite research. With these tools, research could bet-
ter determine the need for and impact of enrichment for
both individual captive welfare and long-term conserva-
tion success in amphibians. Such knowledge could help
to successfully and humanely maintain these animals in
captivity and to successfully release them into the wild.
Acknowledgments. — We are grateful for comments
on the manuscript drafts provided by Silviu Petrovan,
Victoria Ogilvy and Beatrice Gini and for suggestions
from Simon Girling (Head Vet, Royal Zoological Soci-
ety of Scotland).
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Received: 26 March 2014
Accepted: 05 June 2014
Published: 13 June 2014
Christopher Michaels is a doctoral student at the University of Manehester. His researeh foeuses on am-
phibian ex situ eonservation and partieularly on the development of empirieally-based husbandry methods.
He gained a First Class BA in Biologieal Seienees from the University of Oxford in 2010 and has a lifelong
faseination with amphibian biology, eonservation, and eaptive husbandry.
Roger Downie is a semi-retired professor of Zoologieal Edueation at the University of Glasgow. Mueh of
his researeh is on the reproduetive eeology of frogs in Trinidad and Tobago. He has teaehing and researeh
interests in bioethies and wildlife eonservation, whieh eome together in eonsidering the welfare of amphib-
ians.
Roisin Campbell-Palmer is presently the Conservation Projeets Manager for the Royal Zoologieal Soei-
ety of Seotland, where she has worked for 12 years in varying roles beginning as a reptile keeper. For the
last frve years Roisin’s main duties have foeused on the reintroduetion of beavers to Seotland, in her role as
the Field Operations Manager for the Seottish Beaver Trial and undertaking her Ph.D. in beaver health and
welfare through Telemark University College, Norway. Roisin eompleted her honours degree in Zoology
at the University of Glasgow and her MSe in Applied Animal Behaviour and Welfare at the University of
Edinburgh. She is passionate about native wildlife eonservation.
Amphib. Reptile Conserv. | amphibian-reptile-conservation.org (23)
June 2014 I Volume 8 I Number 1 I ell
Amphibian & Reptiie Conservation
[General Section] 8(1): 24-32.
Copyright: © 2014 Mendon^a et al. This is an open-access article distributed under the
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License, which permits unrestricted use for non-commercial and education purposes only
provided the original author and source are credited. The official publication credit source:
Amphibian & Reptile Conservation at: amphibian-reptile-conservation.org
Caatinga Ethnoherpetology: Relationships between herpeto-
fauna and peopie in a semiarid region of northeastern Brazii
^’^Livia Emanuelle Tavares Mendonga, ^Washington Luiz Silva Vieira, and
^’ Romuio Romeu Nobrega Aives
^•^Departamento de Biologia, Universidade Estadual da Paraiba, Av. das Baraunas, 351/Campus Universitdrio Bodocongo, 58109-753 Campina
Grande, PB, BRAZIL Departamento de Sistemdtica e Ecologia da Universidade Eederal da Paraiba, Programa de Pds-Graduagdo em Ciencias
Biologicas (Zoologia), Laboratorio de Ecofisiologia Animal, 58051-900 Jodo Pessoa, PB, BRAZIL
Abstract . — ^We investigated the interactions between humans and herpetofauna in the semiarid
region of Paraiba State, Brazii. Data were obtained by means of interviews with 124 hunters or
ex-hunters using semi-structured questionnaires, compiemented by informai conversations. We
recorded 18 species (17 reptiies and one amphibian) that iocai human popuiations interact with
because they have some utiiitarian vaiue or because of confiicting reiations with iocai inhabitants.
Impiementation of conservation measures aimed at the herpetofauna in this region is particuiariy
difficuit due to the aversion that iocai peopie hoid toward many of these species. Therefore,
environmentai education strategies shouid be adopted. These efforts shouid not be soieiy directed
at species subject to hunting, but shouid be aii-inciusive and take into consideration the cuiturai,
sociai, and utiiitarian roie that governs the interactions of human popuiations and the herpetofauna
of the Caatinga.
Key words. Caatinga, conservation, ethnobiology, ethnozoology, hunting, reptiles, wildlife use
Citation: Mendonga LET, Vieira WLS, Alves RRN. 2014. Caatinga Ethnoherpetology: Relationships between herpetofauna and people in a semiarid region
of northeastern Brazil. Amphibian & Reptile Conservation 8(1) [General Section]: 24-32 (e78).
Introduction
Humans and herpetofauna (amphibians and reptiles)
have interacted for millennia, virtually wherever they
have been in contact (Alves et al. 2013b). As a result,
interactions between humans and these animals are quite
varied, encompassing utilitarian, symbolic, and conflict-
ing aspects (Alves et al. 2008, 2009a, 2012b, c; Fer-
nandes-Ferreira et al. 2012a; Franke and Telecky 2001;
Klemens and Thorbjamarson 1995; Morris and Morris
1965; Moura et al. 2010; Schlaepfer et al. 2005). Such
interactions can be studied through ethnoherpetology,
a subdivision of ethnozoology, which examines the re-
lationships between human cultures and herpetofauna
(Bertrand 1997; Das 1998; Goodman and Hobbs 1994;
Speck 1946). Ethnozoological studies can aid in the
evaluation of the impacts human populations have on na-
tive animal species and in the development of sustainable
management plans, and thus, they are essential to conser-
vation efforts (Alves 2012; Alves and Souto 2011).
Caatinga is the name given to the semiarid region that
occupies the largest portion of Northeast Brazil and rep-
resents one of the major examples of a semiarid environ-
ment in the Neotropical region (Albuquerque et al. 2012;
Alves et al. 2012b). In this biome, 205 herpetofaunal
species have been recorded (65 amphibians, 66 lizards,
12 amphisbaenids, 53 snakes, flve testudines, and four
crocodilians), many of which interact with local human
populations, where they furnish products exploited by
the local people or are hunted and killed due to conflict-
ing relations with people (Alves et al. 2009b, 2012a, b,
c; Barbosa et al. 2011; Femandes-Ferreira et al. 2013). In
this context, understanding of the relations between hu-
mans and the herpetofauna of the region is an important
step in designing strategies for management and sustain-
able use, and should consider the ecological, economic,
and cultural aspects associated with these interactions.
Ethnoherpetological studies have only recently begun
in Caatinga, although general ethnozoological research
indicates that reptiles and amphibians are hunted by ru-
ral and urban populations of the region (Albuquerque et
al. 2012; Alves et al. 2012b; Eernandes-Eerreira et al.
2012a). In an effort to contribute to our ethnoherpetologi-
cal knowledge and its implications in the semiarid region
of northeastern Brazil, we investigated the interactions
between humans and herpetofauna in the municipality of
Pocinhos in the semi-arid region of Paraiba State (PB).
Our aim was to record the patterns of interactions of the
local people with representatives of this animal group in
the region. This information may be used to enhance con-
servation of the Caatinga’ s herpetofauna.
Correspondence. Email: romulo_nobrega@yahoo.com.br
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Mendonga et al.
Materials and Methods
Study area
The present study was carried out in the municipality
of Pocinhos, located in the semi-arid region of Paraiba
State, Brazil (Fig. 1 ; Ribeiro 2003). Pocinhos is 630 km^
in area, with approximately 17,032 inhabitants. Average
annual temperature is 23 °C, which varies little through-
out the year. The region has a very low rainfall rate, fluc-
tuating annually between 400 and 600 mm. The climate
is hot, semi-arid, with rainfall in the autumn and winter
months (Ribeiro 2003) and the vegetation is dominated
by sub-deciduous and deciduous forests typical of semi-
arid regions (Alves et al. 2009b; Ribeiro 2003).
Procedures
The study was conducted in the period of June 2010 to
June 2011. The information was obtained by means of
interviews with hunters or ex-hunters using semistruc-
tured questionnaires, complemented by informal con-
versations (Bernard 1994; Huntington 2000). The selec-
tion of informants was done by the “snowball” sampling
technique (Bailey 1994), where from the initial contact,
an informant indicates another who in turn indicates still
another and so forth. Before each interview, the nature
and objectives of the research were explained, and the
interviewees gave their permission to record the informa-
tion, by signing an informed consent form.
The questionnaires were applied to 124 hunters from
the municipality, of which 98 (79%) live in urban ar-
eas but frequently travel to rural areas to hunt, while 26
(21%) live in the rural zone. The ethical approval for the
study was obtained from the Ethics committee of Hospi-
tal Lauro Wanderley (protocol number: CEP/HULW n°
103/10).
Vernacular names of the specimens cited were re-
corded and the animals identified in the following ways:
(1) analysis of the specimens or parts thereof donated by
the interviewees; (2) analysis of photographs of animals
taken during the interviews and during the accompani-
ment of hunting activities; (3) use of identifications by
taxonomists familiar with the fauna of the study area and
use of vernacular names; and (4) information from previ-
ous ethnozoological studies carried out in the study area
(Alves et al. 2009b; Confessor et al. 2009; Mendonga et
al. 2011). The scientific nomenclature of the species that
are cited in this study follows the guidelines of the Bra-
zilian Society of Herpetology (http://www.sbherpetolo-
gia.org.br/).
After analysis, specimens were deposited at the zoo-
logical collections of the Universidade Federal da Parai-
ba. Samples were collected with the permission of the In-
stitute Chico Mendes de Conservagao da Biodiversidade
(ICMBio) and the Sistema de Autorizagao e Informagao
em Biodiversidade (SISBIO), license number 25926-2.
Data Analysis
An accumulation curve of the herpetofaunal species cited
by interviewees was prepared. In an accumulation curve
for ethnobiological data, the X-axis corresponds to the
number of individuals interviewed and Y-axis the num-
ber of species cited by the respondents. The curve was
randomized 1,000 times and the means were calculated
using the software Estimates© version 8.2 (Colwell
2009). Estimates© permits the statistical analysis of spe-
cies richness (for this work, species richness can be in-
terpreted as the richness of species locally exploited) of
samples by determination of the Chao2 index (Colwell
and Coddington 1994). This index has been used in pre-
B0'0'0"w 6(ii»0'0"w 40*0'0"W 38*0‘0'‘W 36*0*0"W
Fig. 1. Location of the municipality of Pocinhos (Paraiba State, Northeast Brazil), where the study was conducted.
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Ethnoherpetology of the Caatinga region of Brazil
vious ethnozoological studies (Ferreira et al. 2012; Souto
et al. 2011; Whiting et al. 2011).
The non-parametric estimator Chao2 (Chao 1987) is
calculated by the following formula:
Chao2 — Sobs + f— )
\2MJ
where: Sobs corresponds to the number of species in
a given sample, L is the number of species in only one
sample (“uniques”), and M is the number of species
that occur exactly in two samples. The utilization of the
Chao2 estimator is recommended for ethnozoological
studies since it is a non-parametric estimator based on
data of incidence.
The data were entered in Estimates© using a spread-
sheet of type of respondent (rows) x type of species (col-
unms). In preparing the spreadsheet, a value of 1 was
given for each species mentioned by an interviewee and
0 for those that were not recorded.
For each species we calculated the Use- Value [adapt-
ed from the proposal of Phillips et al. (1994)], a quantita-
tive method that demonstrates the relative importance of
species known locally. This value was calculated using
the following formula: UV = X U/n, where: UV = Use-
Value of the species; U = number of citations per species;
n = number of informants. The calculations of the Use-
Values of any species is based objectively on the impor-
tance attributed by the informants themselves, and does
not depend on the opinion of the researcher.
Results
We recorded 18 species of herpetofauna (17 reptiles and
1 amphibian) that interacted with people in the surveyed
area, either because they have some utilitarian value or
because they are involved in conflicting relations with
local inhabitants (Table 1). Products derived from her-
petofauna were used for the following purposes: food {n
= 1 species), medicinal use {n = 1 species), pets {n = A
species), ornamental use {n = A species), and commerce
{n = 2 species). Additionally, 13 species were hunted be-
cause they are considered harmful (particularly snakes),
although some of these also provide products of utilitar-
ian value.
Based on the data collected, the mean number of spe-
cies observed (Sobs) was compared with that expected to
be cited in the surveyed area (Fig. 2). The results demon-
strated that the sampling efficiency was adequate, since
78.4% of all species of the herpetofauna of ethnozoologi-
cal importance for the study area (Chao2 = 22.96 + 5.07)
were recorded. The species accumulation curve showed
a tendency to stabilize.
When we considered the utilitarian value of the herpe-
tofauna in the area studied, a greater number of species
were cited for their utilization as food {n = 1 species),
where lizards were the principal group cited for this pur-
pose, mainly the White tegu {Salvator merianae, Du-
meril and Bibron 1839; Use- Value = 0.66). Other lizards
reported as being used for food were the Green iguana
{Iguana iguana, Linnaeus 1758) and the whiptail lizard
{Ameivula ocellifera, Spix 1825), with the latter being
rarely consumed, as it was cited by only two interview-
ees. In relation to snakes, only three hunters cited species
useful as food: rattlesnake {Crotalus durissus, Linnaeus
1758) and Rainbow boa {Epicrates assist, Machado
1945). The Northeastern pepper frog {Leptodactylus vas-
tus, Lutz 1930) is the only amphibian used for food ac-
cording to interviewees.
The medicinal use of herpetofauna, reported by 28
hunters, appears to be the most connnon form of utiliza-
tion for this animal group. The species most utilized for
this purpose, according to the interviewees, are the White
tegu {n = 28 citations). Green iguana {n = 14 citations),
and rattlesnake {n = S citations; Table 2). Lrom the ani-
mals cited as useful in popular medicine, various parts
or medicinal subproducts are extracted, especially the
fat and hide, which are used in the treatment of various
diseases and are administered in various ways (Table 2).
Use of reptiles as pets was recorded in only three of the
homes visited, suggesting that the use of herpetofauna as
pets is not a connnon practice in the study area. Species
used as pets were: Red footed tortoise {Chelonoidis car-
bonaria, Spix 1824; raised by three hunters), Tuberculate
toadhead turtle {Mesoclemmys tuberculata, Ltiederwaldt
1926), White tegu, and Boa snake {Boa constrictor, Lin-
naeus 1758; cited by only one hunter). The hunter who
mentioned this last species stated that he captured the
animal by hand on a hunting trip, but that he did not keep
the animal long at his home because he was unable to
feed it adequately, thus letting it go in the forest.
Fig. 2. Graphs showing the values obtained with the richness
estimators of herpetofaunal species hunted in surveyed area.
Number of Species Observed (Sobs =18 + 2.44), Number of
species estimated (Chao2 = 22.96 + 5.07).
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Mendonga et al.
Table 1. Hunted herpetofaunal species with their respective popular names in the surveyed area. Legend: F = food resource, M =
medicinal, C = commerce, P = pets, O = ornamentation and decoration, and CR = conflicting relationships.
Family/species/popular name
Use- Value
Uses and/or conflicting relationships
F
M
c
p
0
CR
Leptodactylidae
Leptodactylus vastus (Lutz, 1930) - “Jia,” Northeastern pepper frog
Testudinidae
0.01
X
X
Chelonoidis carbonaria (Spix, 1824) -“Jabuti,” Red footed tortoise tortoise
0.01
X
X
Chelidae
Mesoclemmys tuberculata (Luederwaldt, 1926) - “Cagado d’agua,” Tuberculate
toadhead turtle
0.008
X
X
Iguanidae
Iguana iguana (Linnaeus, 1758) - “Camaleao,” Common green iguana
Teiidae
0.20
X
X
X
Ameivula ocellifera (Spix, 1825) - “Calango,” Spix’s whiptail
0.008
X
Salvator merianae (Dumeril and Bibron, 1839) - “Teju,” White tegu
0.66
X
X
X
X
X
X
Boidae
Boa constrictor (Linnaeus, 1758) - “Cobra de veado,” “jiboia,” Boa snake
0.03
X
X
X
X
Epicrates assisi (Linnaeus, 1758) - “Salamanta,” Rainbow boa
0.16
X
X
X
Colubridae
Oxybelis aeneus (Wagler, 1824) - “Cobra de cipo,” Brown vine snake
Dipsadidae
0.008
X
Boiruna sertaneja (Zaher, 1996) -“Cobra preta,” Black snake
0.02
X
Philodryas olfersii (Linchtestein, 1823) - “Cobra verde,” Lichtenstein’s Green
racer
0.02
X
Leptodeira annulata (Linnaeus, 1758) - “Jararaca,” Salamanta de parede. Banded
cat-eyed snake
0.11
X
Philodryas nattereri (Steindachner, 1870) - “Cobra corre campo,” Paraguay green
racer
0.04
X
Pseudoboa nigra (Dumeril, Bibron e Dumeril, 1854) - “Cobra de leite,” Black
false boa
0.01
X
Xenodon merremii (Wagler, 1824) - “Jararaquinha,” “Goipeba,” Wagler’s snake
Elapidae
0.01
X
Micrurus ibiboboca (Merrem, 1820) - “Cobra coral,” Caatinga coral snake
Viperidae
0.11
X
Bothrops erythromelas (Amaral, 1923) - “Malha de cascavel,” Jararaca da seca,
Caatinga lancehead
0.02
X
Crotalus durissus (Linnaeus, 1758) - “Cascavel,” South American rattlesnake
0.20
X
X
X
X
X
The use of herpetofauna to make artisanal products
was mentioned by only three interviewees, where the
hide is the principal product used for this purpose. This
product is used mainly in the manufacture of accesso-
ries (belts, purses, and key chains). The species used
for this purpose are: rattlesnake, whose rattle is used in
the manufacture of key chains by some hunters and the
hide, which can be used to make belts; and Boa snake.
Rainbow boa, and White tegu, whose hide is used in the
manufacture of accessories.
Despite being sources of products used for different
purposes, the main motivation for the hunting and killing
of the herpetofauna in the study region is that many of
the species cited are considered harmful, particularly the
snakes, considered venomous and efficient predators that
pose a risk to humans and their domestic animals. Forty
(32.2%) hunters interviewed affirmed having killed some
type of reptile while hunting or during daily activities in
the countryside. Meanwhile, the hunters were unanimous
in stating that they kill whatever snake they encounter.
The most persecuted species are the rattlesnake {n =
26 citations). Rainbow boa {n = 2\ citations), Caatinga
lancehead {Bothrops erythromelas, Amaral 1923; n = 2
citations), and coral snake (Micrurus ibiboboca, Merrem
1820; n= 14 citations).
Besides snakes, the White tegu can be killed by some
hunters (n = 4) of rural areas because they do damage,
since this lizard feeds on chicks and chicken eggs. The
latter are important food for local families, besides being
a source of income when sold.
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Ethnoherpetology of the Caatinga region of Brazil
Table 2. Herpetofauna used for medicinal purposes cited by
hunters in the Pocinhos city, Paraiba State, Brazil.
Species /
vernacular
name
Citations
Medicinal use
(Treated diseases)
Parts
Chelonoidis car-
bonaria (Spix,
1824)
2
Rheumatism and
swelling
Shell
and fat
Sore throat, cough,
Mesoclemmys
asthma, earache,
tuberculata
0
wounds, rheuma-
Fat
(Luederwaldt,
tism, haemorrhoids,
1926)
shortness of breath,
bronchitis
Suck a splinter out
of skin or flesh,
Skin,
fat, and
bone
Iguana iguana
14
snakebite, choking,
(Linnaeus, 1758)
boils, rheumatism,
earache, sore throat,
and wounds
Sore throat, earache,
choking, deafness,
Salvator meri-
anae (Dumeril e
Bibron, 1839)
28
boils, wounds, arthri-
tis, asthma, rheuma-
tism, headache, tumor;
suck a splinter out of
skin or flesh, cough,
and swelling
Fat,
tongue,
and skin
Asthma, sore throat,
skin problems, cancer,
rheumatism, urinary
Crotalus duris-
problems, arthritis.
sus (Linnaeus,
1758)
8
toothache, haemor-
rhoids, backache,
mycoses, wounds,
deafness, and varicose
veins
and fat
Leptodactylus
vastus (Lutz,
1930)
1
Sore throat
Meat
Discussion
Our results reveal that the people of the surveyed area
establish a greater interaction with reptiles than amphib-
ians. This finding can be related to the greater richness of
reptiles that occurs in the Caatinga (140 reptiles and 65
amphibians) and also among the reptiles there are larger-
sized species, which can offer larger amounts of products
for use. Snakes are feared animals in all of the semiarid
northeast and in other places in Brazil, calling extra at-
tention associated with the prevention of potential acci-
dents (Alves et al. 2010b, 2012b, c; Moura et al. 2010).
Despite the negative view related to the many spe-
cies of reptiles in the area studied, there are many spe-
cies (even those killed because of conflicts) that supply
products used by the local inhabitants. These observa-
tions are in agreement with Marques (1995), who noted
that the link between humans and animals is fraught with
contradictions and ambiguities, as the native fauna can
represent either a resource or a risk to the local people.
The small number of species of herpetofauna used as
food is not surprising, since traditionally, this group does
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not play an important role as a protein source for the pop-
ulations living in the Caatinga. The principal groups of
wild vertebrates used as a source of protein in the region
are birds and mancunals (Alves et al. 2009b; Bezerra et al.
2011, 2012a, b, 2013; Femandes-Ferreira et al. 2012b).
However, the game importance of the White tegu should
be pointed out, as its meat is used as a source of protein
in the Caatinga. Such observation can be substantiated
in a parallel study on the consumption of bushmeat in
the same area as the present study (Mendonga 2012),
which monitored the consumption of meat by local fami-
lies during a year and within the local herpetofauna, only
recording the consumption of two species of reptiles: S.
merianeae and 1. iguana, with greater frequency for the
former. The use of these two species for food also has
been recorded in other localities of the senharid north-
east, including urban areas (Alves et al. 2012a; Marques
and Guerreiro 2007). Considering the cultural and utili-
tarian importance of the lizard S. merianae, we do not ex-
aggerate when we suggest that this animal represents one
of the animals of greatest ethnozoological importance in
the Brazilian Caatinga. This can be due to its size, since
it is the largest species of lizard of the semiarid region
(Vanzolini et al. 1980) and corroborates the findings of
Alves et al. (2012b), which pointed out that S. merianeae
represents the main game reptile of the senh-arid region
of Brazil.
Corroborating a tendency observed in other studies
(Alves et al. 2012c; Marques and Guerreiro 2007; San-
tos-Fita et al. 2010), the consumption of snakes was little
cited by the hunters in the study area. In Brazil, only five
snake species have been reported as being used for hu-
man consumption: Boa constrictor, Eunectes murinus,
Lachesis muta, Crotalus durissus, and Epicrates as-
sist (Alves et al. 2012c; Fernandes-Ferreira et al. 2013).
Alves et al. (2012c) highlighted that the small numbers
of snake species currently used as food in Brazil is not
surprising given the negative images attributed to these
animals in myths, legends, and popular beliefs. Reinforc-
ing this notion, Rea (1981) noted that not only are snakes
rejected because of their disagreeable nature but also any
other creature with a similar shape or behavior. A study
undertaken among human populations living along the
banks of the Rio Negro (Amazonas State, Brazil) indi-
cated that the electric eel (Electrophorus electricus) was
one of the least favored meats because of its strong smell
and the shape of its body — “it looks just like a snake”
(Silva 2007).
Although the herpetofauna does not play an impor-
tant role as a source of protein in the region studied, this
group stands out when considering the popular medicine
of the region. Despite having been cited less as medici-
nal species {n = 6 species) than as those used for food
{n = 1 species), medicinal use showed a higher number
of citations, suggesting its greater dissenunation among
the interviewees. In this context, the tegu was also fea-
tured with regard to number of citations as well its broad
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Mendonga et al.
medicinal applicability. Studies in various localities have
already indicated the importance of this species of lizard
in popular medicine in Brazil (Alves 2009; Alves et al.
2007, 2009a, 2011; Ferreira et al. 2012; Oliveira et al.
2010), even in urban areas, where the sale of products
derived from S. merianeae (as well as other species of
reptiles recorded in this work) is common in public mar-
kets in various cities in northeast Brazil (Alves and Rosa
2007; Alves and Rosa 2010; Ferreira et al. 2012).
Raising wild animals as pets, particularly wild birds
(Alves et al. 2010a, 2013a; Bezerra et al. 2001, 2013;
Femandes-Ferreira et al. 2012b; Nobrega et al. 2012)
is a very conunon practice in the semiarid northeastern
region, but few species of the herpetofauna are utilized
for this reason, in accordance with our finding presented
here. Among the reptiles of the Caatinga, the Jabuti (C.
carbonaria) is one of the species of the most popular
pets, probably because it is considered docile and easy to
capture and keep in captivity. Additionally, there is also a
popular belief that its presence helps avoid illnesses such
as bronchitis and asthma (Alves et al. 2009a).
The strong aversion to reptiles, especially snakes, is
conunon in various places in Brazil (Alves et al. 2012b, c;
Moura et al. 2010; Santos-Fita et al. 2010), and was also
recorded in our study. This aversion serves as a strong
motivation for hunters and the public in general to kill
snakes indiscriminately, where they are persecuted and
killed whenever they are encountered. People are used
to killing not only venomous snakes but also the non-
venomous species, and even those amphibians that have
a similar body shape as snakes. Similarly, Santos-Fita et
al. (2010) documented that all inhabitants of a semiarid
area of the state of Bahia have strong negative reactions
in relation to snakes, always killing them if possible. It
should be emphasized that these conflicts involve other
groups besides snakes. In our study, we recorded that
even species of reptiles with high utilitarian value, such
as the tegu, can also be killed for feeding on chicken
eggs, causing financial losses for farmers.
Our data, together with previous findings of other eth-
nozoological studies carried out in the semiarid region of
the northeast, allow us to suggest some patterns of inter-
actions between the people and herpetofauna of the Caat-
inga: (1) there are more frequent interactions between
the people and reptiles than with amphibians; (2) lizards
comprise the group with the most important species for
food, particularly the White tegu; (3) products from her-
petofauna play an important role in popular medicine in
the semiarid, northeastern region; (4) besides food and
medicinal use, products from herpetofauna can be used
in handicrafts and jewelry; and, (5) various reptile spe-
cies, especially snakes, are hunted and killed because of
cultural aversion to these animals and the risks they pose
to people and domestic animals.
Information from previous studies and that obtained
here demonstrate that in the semiarid region of Brazil’s
northeast, reptiles and amphibians are hunted because
Amphib. Reptile Conserv. | amphibian-reptile-conservation.org (29)
they are useful or considered dangerous, and sometimes
for both reasons. The implementation of conservation
measures aimed at the herpetofauna in this region is par-
ticularly difficult due to the aversion of the people to a
good part of the species of this group. Therefore, strate-
gies of environmental education should be adopted, be-
sides specific actions directed at species of high game
value, taking into consideration the cultural, social, and
utilitarian role that governs the interactions of human
populations and the herpetofauna of the Caatinga.
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Received: 17 January 2014
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Published: 05 July 2014
Amphib. Reptile Conserv. | amphibian-reptile-conservation.org (31)
July 2014 I Volume 8 | Number 1 | e78
Ethnoherpetology of the Caatinga region of Brazil
Livia Emanuelle Tavares Mendon^a is a biologist, M.Sc. in Zoology at the Universidade Federal da
Paraiba (UFPB), Bachelor in Biological Sciences from the Universidade Estadual da Paraiba (UEPB). She
develops research focused on hunting, wildlife conservation, and Ethnozoology (Photographed by Romulo
Alves).
Washington Luiz Silva Vieira is a biologist, Ph.D. in Zoology at the Universidade Federal da Paraiba. His
main interests are taxonomy, ecology, and natural history of herpetofauna. He also has research interests in
ethnoherpetology and animal conservation (Photographed by Romulo Alves).
Romulo Romeu Nobrega Alves is a professor at the Universidade Estadual da Paraiba, Brazil, where he
teaches undergraduate and graduate courses in Biological Sciences and Ecology. His Ph.D. (Zoology) was
completed in 2006, at the Universidade Federal da Paraiba. His areas of professional interest are ethnozool-
ogy and wildlife trade, uses and conservation, zootherapy, and human ecology. He has conducted ethno-
biological research for the last ten years in Brazil which focuses on fisheries, hunting, and wildlife trade
and uses. Currently, he coordinates projects on hunting and uses of wildlife in Brazil. In addition, he is one
of the Editors-in-Chief of the journal of Ethnobiology and Conservation and of the Editorial Board of the
Journal of Ethnobiology and Ethnomedicine. Prof. Romulo R. N. Alves holds a Productivity scholarship,
provided by the National Council of Science and Technology (CNPq) (Photographed by Wedson Souto).
Amphib. Reptile Conserv. | amphibian-reptile-conservation.org (32)
July 2014 I Volume 8 | Number 1 | e78
Special Section
CONTENTS
Ryan L. Lynch, Sebastian Kohn, Fernando Ayala-Varela, Paul S. Hamilton, and Santiago R.
Ron — Rediscovery of Andinophryne olallai Hoogmoed, 1985 (Anura, Bufonidae), an enigmatic
and endangered Andean toad 1
Fernando P. Ayala-Varela, Diana Troya-RodrIguez, Xiomara Talero-RodrIguez and Omar Torres-Car-
VAJAL — A new Andean anole species of the Dactyloa clade (Squamata: Iguanidae) from western Ecuador. ... 8
Santiago R. Ron, Andrea E. Narvaez, and Giovanna E. Romero — Reproduction and spawning behav-
ior in the frog, Engystomops pustulatus (Shreve 1941) 25
Juan M. Guayasamin, Angela Maria Mendoza, Ana V. Eongo, Kelly R. Zamudio, and Elisa Bonac-
CORSO — High prevalence of Batrachochytrium dendrobatidis in an Andean frog community (Reserva
Eas Gralarias, Ecuador) 33
Ana Almendariz, John E. Simmons, Jorge Brito, and Jorge Vaca-Guerrero — Overview of the herpeto-
fauna of the unexplored Cordillera del Condor of Ecuador 45
Shawn F. McCracken and Michael R. J. Forstner — Herpetofaunal community of a high canopy tank bro-
meliad {Aechmea zebrind) in the Yasuni Biosphere Reserve of Amazonian Ecuador, with comments on
the use of “arboreal” in the herpetological literature 65
Omar Torres-Carvajal, Pablo J. Venegas, Simon E. Eobos, Paola Mafla-Endara, and Pedro M. Sales
Nunes — A new species of Pholidobolus (Squamata: Gymnophthalmidae) from the Andes of south-
ern Ecuador 76
MarIa-Jose Salazar-Nicholls and Eugenia M. del Pino — Early development of the glass frogs Hyalino-
batrachium fleischmanni and Espadarana callistomma (Anura: Centrolenidae) from cleavage to tadpole
hatching 89
David Salazar-Valenzuela, Angele Martins, Euis Amador-Oyola, and Omar Torres-Carvajal — A
new species and country record of threadsnakes (Serpentes: Eeptotyphlopidae: Epictinae) from northern
Ecuador 107
Francisca Hervas, Karina P. Torres, Paola Montenegro-Earrea, and Eugenia M. del Pino — Develop-
ment and gastrulation in Hyloxalus vertebralis and Dendrobates auratus (Anura: Dendrobatidae) 121
Fernando Ayala-Varela, Julian A. Velasco, Martha Calderon-Espinosa, Alejandro F. Arteaga, Yerka
Sagredo, and Sebastian Valverde — First records of Anolis ventrimaculatus Boulenger, 1911 (Squama-
ta: Iguanidae) in Ecuador 136
Howard O. Clark, Jr. and Craig Hassapakis — ^The Amphibians and Reptiles of Mindo 141
Oscar Angarita-M., Andres Camilo Montes-Correa, and Juan Manuel Renjifo — Amphibians and rep-
tiles of an agroforestry system in the Colombian Caribbean 143
General Section
Todd W. Pierson, Yan Fang, Wang Yunyu, and Theodore Papenfuss — ^A survey for the Chinese gi-
ant salamander {Andrias davidianus; Blanchard, 1871) in the Qinghai Province 1
Christopher J. Michaels, J. Roger Downie, and Roisin Campbell-Palmer — ^The importance of enrichment
for advancing amphibian welfare and conservation goals: A review of a neglected topic 7
EiviA Emanuelle Tavares Mendon^a, Washington Euiz Silva Vieira, and Romulo Romeu Nobrega
Alves — Caatinga Ethnoherpetology: Relationships between herpetofauna and people in a semiarid region
of northeastern Brazil 24
Table of Contents Back cover
Cover: An adult individual of the Tandayapa Andean Toad (Rhaebo olallai, syn. Andinophryne olallai) from an extant population discovered
in Reserva Rio Manduriacu, Imbabura Province, Ecuador. Prior to the discovery of the population in Reserva Rio Manduriacu, the species had
not been documented since its original description in 1970 and was presumed Extinct. The rediscovery of the species in 2014 resulted in the col-
lection of the first color photographs of the species, information on its ontogeny and natural history, the species conservation status, and led to a
taxonomic update of the genus Andinophryne. Photography: Ryan L. Lynch.
Instructions for Authors: Eocated at the Amphibian & Reptile Conservation website:
http://amphibian-reptile-conservation.org/submissions.html
Copyright: © 2014 Craig Hassapakis/Amphibian & Reptile Conservation
VOLUME 9 2014
NUMBER 1
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